Subtilisin carlsberg proteins with reduced immunogenicity

ABSTRACT

The present invention provides methods for the identification of CD4 +  T-cell epitopes in subtilisin Carlsberg proteins. The present invention also provides for the production of altered peptides which, when incorporated into a wild-type subtilisin Carlsberg protein produce an altered immunogenic response, preferably a low immunogenic response in humans. In particular, the present invention provides means, including methods and compositions suitable for reducing the immunogenicity of ALCALASE® enzyme.

FIELD OF THE INVENTION

The present invention provides methods for the identification of CD4⁺T-cell epitopes in subtilisin Carlsberg proteins. The present inventionalso provides for the production of altered peptides which, whenincorporated into a wild-type subtilisin Carlsberg protein produce analtered immunogenic response, preferably a low immunogenic response inhumans. In particular, the present invention provides means, includingmethods and compositions suitable for reducing the immunogenicity ofALCALASE® enzyme.

BACKGROUND OF THE INVENTION

Proteins used in industrial, pharmaceutical and commercial applicationsare of increasing prevalence and importance. However, this has resultedin the sensitization of numerous individuals to these proteins,resulting in the widespread occurrence of allergic reactions to theseproteins. For example, some proteases are associated withhypersensitivity reactions in certain individuals. As a result, despitethe usefulness of proteases in industry (e.g., in laundry detergents,cosmetics, textile treatment etc.), as well as the extensive researchperformed in the field to provide improved proteases (e.g., variantsubtilisin Carlsberg enzymes with more effective stain removal undertypical laundry conditions), the use of proteases in industry has beenproblematic.

Much work has been done to alleviate these problems. Strateg iesexplored to reduce immunogenic potential of protease use includeimproved production processes which reduce potential contact bycontrolling and minimizing workplace concentrations of dust particlesand/or aerosol carrying airborne protease, improved granulationprocesses which reduce the amount of dust or aerosol actually producedfrom the protease product, and improved recovery processes to reduce thelevel of potentially allergenic/immunogenic contaminants in the finalproduct. However, efforts to reduce the allergenicity/immunogenicity ofproteases themselves have been relatively unsuccessful. Alternatively,efforts have been made to mask epitopes in protease which are recognizedby immunoglobulin E (IgE) in hypersensitive individuals (See, PCTPublication No. WO 92/10755; WO 94/10191; WO 96/17929; WO 99/49056; andWO 01/07578), or to enlarge or change the nature of the antigenicdeterminants by attaching polymers or peptides/proteins to theproblematic protease.

While some studies have provided methods of reducing theallergenicity/immunogenicity of certain proteins and identification ofepitopes which cause allergic reactions in some individuals, the assaysused to identify these epitopes generally involve measurement of IgE andIgG in the sera of those who have been previously exposed to theantigen. However, once an Ig reaction has been initiated, sensitizationhas already occurred. Accordingly, there is a need to identify proteinswhich produce an enhanced immunologic response, as well as a need toproduce proteins which produce a reduced immunologic response.

SUMMARY OF THE INVENTION

The present invention provides methods for the identification of CD4⁺T-cell epitopes in subtilisin Carlsberg proteins. The present inventionalso provides for the production of altered peptides which whenincorporated into a wild-type subtilisin Carlsberg protein produce analtered immunogenic response, preferably a low immunogenic response inhumans. In particular, the present invention provides means, includingmethods and compositions suitable for reducing the immunogenicity ofALCALASE® enzyme. The present invention also provides for the productionof altered peptides which when incorporated into a wild-type subtilisinCarlsberg protein sequence, are no longer capable of initiating the CD4⁺T-cell response or at least reduce the allergic response. In particular,the present invention provides means, including methods and compositionssuitable for reducing the immunogenicity of a wild-type subtilisinCarlsberg.

In one embodiment, the present invention provides T-cell epitopes of theALCALASE® enzyme. These epitopes are provided in various sequences setforth herein (See, FIGS. 2 and 3) and include but are not limited topeptide number 5 (SEQ ID NO. 6); peptide number 26 (SEQ ID NO: 27);peptide number 37 (SEQ ID NO: 38); peptide number 50 (SEQ ID NO: 51);peptide number 51 (SEQ ID NO: 52) and peptide number 79 (SEQ ID NO: 80).In another embodiment, the present invention provides altered sequencesof the identified epitopes which are suitable for substitution into theALCALASE® enzyme.

In a further embodiment, the present invention provides assay systemsfor identification of T-cell epitopes and T-cell non-epitopes, includingbut not limited to methods having the steps of combining differentiateddendritic cells with human CD4⁺ and/or CD8⁺ T-cells and with a peptideof interest (e.g., peptides derived from the ALCALASE® enzyme). Morespecifically, peptides of interest that produce a reduced immunogenicresponse are provided, wherein a T-cell epitope is recognized comprisingthe steps of: (a) obtaining from a single blood source a solution ofdendritic cells and a solution of CD4⁺ and/or CD8⁺ T-cells; (b)promoting differentiation of the dendritic cells; (c) combining thesolution of differentiated dendritic cells, CD4⁺ T-cells and/or CD8⁺T-cells with a peptide of interest (e.g., a peptide comprising at leasta portion of the ALCALASE®); and (d) measuring the proliferation of theT-cells in step (c). (See e.g., WO99/53038; and Strickler et al., J.Immunother., 23:654-660 [2000]).

In yet another embodiment of the invention, a series of peptideoligomers which correspond to all or parts of the ALCALASE® enzyme areprovided. For example, a peptide library is produced covering therelevant portion or all of the ALCALASE® enzyme. In one aspect, themanner of producing the peptides is to introduce overlap into thepeptide library, for example, producing a first peptide whichcorresponds to amino acid sequence 1-15 of the ALCALASE® enzyme, asecond peptide which corresponds to amino acid sequence 4-18 of theALCALASE® enzyme, a third peptide which corresponds to amino acidsequence 7-21 of the ALCALASE® enzyme, a fourth peptide whichcorresponds to amino acid sequence 10-24 of the ALCALASE® enzyme, etc.until representative peptides corresponding to the entire ALCALASE®enzyme molecule are created. By analyzing each of the peptidesindividually in the assay provided herein, it is possible to preciselyidentify the location of epitopes recognized by T-cells. In the exampleabove, the greater reaction of one specific peptide as compared to itsneighbors facilitates identification of the epitope anchor region towithin three amino acids. After determining the location of theseepitopes, it is possible to alter one or more of the amino acids withineach epitope. The modified epitope(s) may then be reincorporated intothe backbone of the wild-type subtilisin Carlsberg protein. Inparticularly preferred embodiments, the resulting modified subtilisinCarlsberg proteins produces a different T-cell response, preferably areduced T-cell response, as compared to that produced by the originalwild-type protein. Moreover, the present invention provides means forthe identification of proteins which naturally have desired low T-cellepitope potency and may find use in their naturally occurring forms.

Various in vitro and in vivo assays known in the art may be used toascertain the reduced immunogenic response of modified proteinsaccording to the invention. In vivo assays include, but are not limitedto HLA class 11 responses and more specifically to HLA-DR3/DQ2 mouseT-cell responses. Suitable in vitro assays include, but are not limitedto human peripheral blood mononuclear cell (PBMC) assays (See e.g.,Herman et al., J. Immunol., 163:6275-6282 [1999]; Sonderstrup et al.,Immunol. Rev., 172: 335-343 [1999]; Taneja and David, Immunol. Rev.,169:67-79 [1999]; Taurog et al., Immunol. Rev. 169:209-223 [1999];Cosgrove, et al., Cell 66:1051-1066 [1991]; and Grusby et al., Proc.Natl. Acad. Sci., 90:3913-3917 [1993]).

The present invention further provides modified subtilisin Carlsbergcompositions with reduced immunogenicity. In particular, the presentinvention provides ALCALASE® enzyme compositions that comprise epitopesdescribed herein that reduce the immunogenic response to the ALCALASE®enzyme.

The present invention also provides methods for identifying at least oneT-cell epitope of a microbial subtilisin, comprising the steps of: (i)obtaining from a from a single human blood source, a solution ofdendritic cells and a solution of naïve CD4+ and/or CD8+ T-cells; (ii)differentiating the dendritic cells to produce a solution ofdifferentiated dendritic cells; (iii) combining the solution ofdifferentiated dendritic cells and naïve CD4+ and/or CD8+ T-cells withpeptide fragments of the subtilisin Carlsberg; and (iv) measuringproliferation of the T-cells in step (iii). In some preferredembodiments, the microbial subtilisin Carlsberg is derived from a memberof the genus Bacillus. In particularly preferred embodiments, theBacillus is selected from the group consisting of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.In alternative preferred embodiments, microbial subtilisin Carlsbergcomprises at least a portion of the sequence set forth in SEQ ID NO:1.

The present invention further provides methods for reducing theimmunogenicity of a microbial subtilisin Carlsberg, comprising the stepsof: (a) identifying at least one T-cell epitope in the protein by (i)contacting an adherent monocyte-derived dendritic cell that has beendifferentiated by exposure to at least one cytokine in vitro, with atleast one peptide comprising a T-cell epitope; and (ii) contacting thedendritic cell and peptide with a naïve T-cell, wherein the naïve T-cellhas been obtained from the same source as the adherent monocyte-deriveddendritic cell, and whereby the T-cell proliferates in response to thepeptide; and (b) modifying the subtilisin Carlsberg to neutralize theT-cell epitope to produce a variant protein, such that the variantprotein induces less than or substantially equal to the baselineproliferation of the naïve T-cells. In some preferred embodiments, themicrobial subtilisin Carlsberg is derived from a member of the genusBacillus. In particularly preferred embodiments, the Bacillus isselected from the group consisting of B. subtilis, B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.coagulans, B. circulans, B. lautus, and B. thuringiensis. In alternativepreferred embodiments, microbial subtilisin Carlsberg comprises at leasta portion of the sequence set forth in SEQ ID NO:1. In some embodiments,the epitope of the microbial subtilisin Carlsberg is modified bysubstituting the amino acid sequence of the T-cell epitope with ananalogous sequence from a homolog of the microbial subtilisin, whereinthe substitution substantially mimics the major tertiary structureattributes of the T-cell epitope. In alternative embodiments, themicrobial subtilisin Carlsberg is modified by altering at least oneepitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:90,SEQ ID NO:15, SEQ ID NO:30, and SEQ ID NO:40. In still furtherembodiments, the epitope is modified by substituting an amino acidsequence for a residue corresponding to at least one of the epitopes. Inyet additional embodiments, the epitope is modified by deleting an aminoacid sequence for a residue corresponding to at least one of theepitopes. In alternative embodiments, the epitope is modified by addingan amino acid to at least one of the epitopes.

The present invention also provides modified subtilisin Carlsbergenzymes (i.e., variant subtilisin Carlsberg enzymes). In some preferredembodiments, these variant subtilisin Carlsberg enzymes comprise atleast one alteration in at least one epitope comprising an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:90, SEQ ID NO:15, SEQ ID NO:30, and SEQ ID NO:40. Indeed, the presentinvention provides numerous variant subtilisin Carlsberg enzymes. Inparticularly preferred embodiments, these variant enzymes exhibitreduced immunogenicity/allergenicity as compared to wild-type subtilisinCarlsberg enzymes. These variant enzymes find use in numerous productsand methods, ranging from personal/consumer care items to industrialproduction and use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the mature protein sequence of subtilisin Carlsberg asfound in the ALCALASE® enzyme (SEQ ID NO: 1). In this Figure, the maturepolypeptide start at position 106 is indicated by the underlinedresidues (AQTVP).

FIG. 2 provides the amino acid sequences of peptide numbers 1-88corresponding to SEQ ID NOS: 2-89, respectively, of the peptidessynthesized based on the sequence of SEQ ID NO: 1.

FIG. 3 provides the assay results for the peptides set forth in FIG. 2.

DESCRIPTION OF THE INVENTION

The present invention provides methods for the identification of CD4⁺T-cell epitopes in subtilisin Carlsberg proteins. The present inventionalso provides for the production of altered peptides which, whenincorporated into a wild-type subtilisin Carlsberg protein produce analtered immunogenic response, preferably a low immunogenic response inhumans. In particular, the present invention provides means, includingmethods and compositions suitable for reducing the immunogenicity ofALCALASE® enzyme. The present invention also provides for the productionof altered peptides which when incorporated into a wild-type subtilisinCarlsberg protein sequence, are no longer capable of initiating the CD4⁺T-cell response or at least reduce the allergic response. In particular,the present invention provides means, including methods and compositionssuitable for reducing the immunogenicity of a wild-type subtilisinCarlsberg.

Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham,The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, the preferred methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the Specification as a whole. Also,as used herein, the singular “a”, “an” and “the” includes the pluralreference unless the context clearly indicates otherwise.

As used herein, “the genus Bacillus” includes all members known to thoseof skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.It is recognized that the genus Bacillus continues to undergotaxonomical reorganization. Thus, it is intended that the genus includespecies that have been reclassified, including but not limited to suchorganisms as B. stearothermophilus, which is now named “Geobacillusstearothermophilus.” The production of resistant endospores in thepresence of oxygen is considered the defining feature of the genusBacillus, although this characteristic also applies to the recentlynamed Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus,Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus,Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, andVirgibacillus.

The term “wild-type subtilisin Carlsberg” as used herein includes: i)the well-known subtilisin Carlsberg proteins such as the ALCALASE®enzyme commercially available from Novo (SEQ ID NO: 1); MAXATASE®enzyme, commercially available from Genencor; and OPTIMASE® enzyme,commercially available from Kali-Chemie; ii) subtilisin Carlsbergproteins having similar catalytic activity to ALCALASE® and having atleast about 80%, 85%, 90%, 95%, 97%, 98% or 99% amino acid sequenceidentity to SEQ ID NO: 1, and preferably at least 90% amino acididentity to SEQ ID NO: 1; and iii) variants of subtilisin Carlsbergproteins.

The term “ALCALASE®” as used herein refers to the serine protease,subtilisin Carlsberg derived from Bacillus licheniformis havingSwissProt Accession number P00780. The total protein has 379 amino acidresidues. The preprotein includes 105 amino acid residues and the matureprotein comprises 274 amino acid residues (See, FIG. 1, SEQ ID NO: 1;See also, Jacobs et al., Nucleic Acids Res., 13: 8913-8926 [1985]; andSmith et al., J. Biol. Chem., 243: 2184-2191 [1968]).

As used herein, the terms “modified wild-type subtilisin Carlsberg”;“modified ALCALASE®” and “modified variant” refer to a protein in whichat least one significant T-cell epitope of the wild-type subtilisinCarlsberg and particularly ALCALASE® has been altered.

As used herein, “wild-type” and “native” proteins are those found innature. The terms “wild-type sequence,” and “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project. The genes encoding thenaturally-occurring (i.e., precursor) protein may be obtained in accordwith the general methods known to those skilled in the art. The methodsgenerally comprise synthesizing labeled probes having putative sequencesencoding regions of the protein of interest, preparing genomic librariesfrom organisms expressing the protein, and screening the libraries forthe gene of interest by hybridization to the probes. Positivelyhybridizing clones are then mapped and sequenced.

“Recombinant,” “recombinant subtilisin” and “recombinant protease” referto a protein, subtilisin or protease in which the DNA sequence encodingthe protein, subtilisin or protease is modified to produce a variant (ormutant) DNA sequence which encodes the substitution, deletion orinsertion of one or more amino acids in the naturally-occurring aminoacid sequence. Suitable methods to produce such modification, and whichmay be combined with those disclosed herein, include, but are notlimited to those disclosed in U.S. Pat. No. 4,760,025 (U.S. RE 34,606),U.S. Pat. No. 5,204,015 and U.S. Pat. No. 5,185,258, all of which areincorporated herein by reference.

“Non-human subtilisins” and the DNA sequences encoding them are obtainedfrom many prokaryotic and eukaryotic organisms. Suitable examples ofprokaryotic organisms include Gram-negative organisms (e.g., E. coli andPseudomonas sp.), as well as Gram-positive bacteria (e.g., Micrococcussp. and Bacillus sp.). Examples of eukaryotic organisms from whichsubtilisins and their genes may be obtained include fungi such asSaccharomyces cerevisiae and Aspergillus sp.

The term “sample” as used herein is used in its broadest sense. However,in preferred embodiments, the term is used in reference to a sample(e.g., an aliquot) that comprises a peptide (e.g., a peptide within apepset, that comprises a sequence of a protein of interest) that isbeing analyzed, identified, modified, and/or compared with otherpeptides. Thus, in most cases, this term is used in reference tomaterial that includes a is protein or peptide that is of interest.

As used herein, “background level” and “background response” refer tothe average percent of responders to any given peptide in the datasetfor any tested protein. This value is determined by averaging thepercent responders for all peptides in the set, as compiled for all thetested donors. As an example, a 3% background response would indicatethat on average there would be three positive (SI greater than 2.95)responses for any peptide in a dataset when tested on 100 donors.

As used herein, “antigen presenting cell” (“APC”) refers to a cell ofthe immune system that presents antigen on its surface, such that theantigen is recognizable by receptors on the surface of T-cells. Antigenpresenting cells include, but are not limited to dendritic cells,interdigitating cells, activated B-cells and macrophages.

As used herein, the terms “T lymphocyte” and “T-cell,” encompass anycell within the T lymphocyte lineage from T-cell precursors (includingThyl positive cells which have not rearranged the T cell receptor genes)to mature T cells (i.e., single positive for either CD4 or CD8, surfaceTCR positive cells).

As used herein, “CD4⁺ T-cell” and “CD4 T-cell” refer to helper T-cells,while “CD8⁺ T-cell” and CD8 T-cell” refer to cytotoxic T-cells.

“T-cell proliferation,” as used herein, refers to the number of T-cellsproduced during the incubation of T-cells with the antigen presentingcells, with or without the presence of antigen.

“Baseline T-cell proliferation,” as used herein, refers to the degree ofT-cell proliferation that is normally seen in an individual in responseto exposure to antigen presenting cells in the absence of peptide orprotein antigen. For the purposes herein, the baseline T-cellproliferation level is determined on a per sample basis for eachindividual as the proliferation of T-cells in response to antigenpresenting cells in the absence of antigen.

As used herein, “T-cell epitope” refers to a feature of a peptide orprotein which is recognized by a T-cell receptor in the initiation of animmunogenic response to the peptide comprising that antigen (i.e., theimmunogen). Although it is not intended that the present invention belimited to any particular mechanism, it is generally believed thatrecognition of a T-cell epitope by a T-cell is via a mechanism whereinT-cells recognize peptide fragments of antigens which are bound to ClassI or Class II MHC (i.e., HLA) molecules expressed on antigen-presentingcells (See e.g., Moeller, Immunol. Rev., 98:187 [1987]).

As used herein, “altered T-cell epitope,” refers to an epitope aminoacid sequence which differs from the precursor peptide or peptide ofinterest, such that the variant peptide of interest produces differentimmunogenic responses in a human or another animal. It is contemplatedthat an altered immunogenic response encompasses altered immunogenicityand/or allergenicity (i.e., an either increased or decreased overallimmunogenic response). In some embodiments, the altered T-cell epitopecomprises substitution and/or deletion of an amino acid selected fromthose residues within the identified epitope. In alternativeembodiments, the altered T-cell epitope comprises an addition of one ormore residues within the epitope.

As used herein, a “weakly significant T-cell epitope” refers to anepitope wherein the response rate within the tested donor pool isgreater than the background response rate, but less than three times thebackground rate.

As used herein, the terms “B lymphocyte” and “B-cell” encompasses anycell within the B-cell lineage from B-cell precursors, such aspre-B-cells (B220⁺ cells which have begun to rearrange Ig heavy chaingenes), to mature B-cells and plasma cells.

As used herein, “B-cell proliferation,” refers to the number of B-cellsproduced during the incubation of B-cells with the antigen presentingcells, with or without the presence of antigen.

As used herein, “baseline B-cell proliferation,” as used herein, refersto the degree of B-cell proliferation that is normally seen in anindividual in response to exposure to antigen presenting cells in theabsence of peptide or protein antigen. For the purposes herein, thebaseline B-cell proliferation level is determined on a per sample basisfor each individual as the proliferation of B-cells in the absence ofantigen.

As used herein, “B-cell epitope,” refers to a feature of a peptide orprotein which is recognized by a B-cell receptor in the immunogenicresponse to the peptide comprising that antigen (i.e., the immunogen).

As used herein, “altered B-cell epitope,” refers to an epitope aminoacid sequence which differs from the precursor peptide or peptide ofinterest, such that the variant peptide of interest produces different(i.e., altered) immunogenic responses in a human or another animal. Itis contemplated that an altered immunogenic response encompasses alteredimmunogenicity and/or allergenicity (i.e., an either increased ordecreased overall immunogenic response). In some embodiments, thealtered B-cell epitope comprises substitution and/or deletion of anamino acid selected from those residues within the identified epitope.In alternative embodiments, the altered B-cell epitope comprises anaddition of one or more residues within the epitope.

As used herein, the term “significant epitope” refers to an epitope(i.e., a T-cell or B-cell epitope) wherein the response rate within thetested donor pool is equal to or greater than about three times thebackground response rate.

As used herein “altered immunogenic response,” refers to an increased orreduced immunogenic response. Proteins and peptides exhibit an“increased immunogenic response” when the T-cell and/or B-cell responsethey evoke is greater than that evoked by a parental (e.g., precursor)protein or peptide (e.g., the protein of interest). The net result ofthis higher response is an increased antibody response directed againstthe variant protein or peptide. Proteins and peptides exhibit a “reducedimmunogenic response” when the T-cell and/or B-cell response they evokeis less than that evoked by a parental (e.g., precursor) protein orpeptide. In preferred embodiments, the net result of this lower responseis a reduced antibody response directed against the variant protein orpeptide. In some preferred embodiments, the parental protein is awild-type protein or peptide.

As used herein, an “in vivo reduction in immunogenicity” refers to anexhibited decrease in the immunogenic response as determined by an assaythat occurs at least in part, within a living organism, (e.g., requiresthe use of an living animal). Exemplary “in vivo” assays includedetermination of altered immunogenic responses in mouse models.

As used herein, an “in vitro reduction in immunogenicity” refers anexhibited decrease in the immunogenic response as determined by an assaythat occurs in an artificial environment outside of a living organism(i.e., does not require use of a living animal). Exemplary in vitroassays include testing the proliferative responses by human peripheralblood mononuclear cells to a peptide of interest.

As used herein, “protein of interest,” refers to a protein (e.g.,protease) which is being analyzed, identified and/or modified.Naturally-occurring, as well as recombinant proteins find use in thepresent invention. Indeed, the present invention finds use with anyprotein against which it is desired to characterize and/or modulate theimmunogenic response of humans (or other animals). In some embodiments,proteins including hormones, cytokines, antibodies, enzymes, structuralproteins and binding proteins find use in the present invention. In someembodiments, hormones, including but not limited to insulin,erythropoietin (EPO), thrombopoietin (TPO) and luteinizing hormone (LH)find use in the present invention. In further embodiments, cytokinesincluding but limited to interferons (e.g., IFN-alpha and IFN-beta),interleukins (e.g., IL-1 through IL-15), tumor necrosis factors (e.g.,TNF-alpha and TNF-beta), and GM-CSF find use in the present invention.In yet other embodiments, antibodies (i.e., immunoglobulins), includingbut not limited to human and humanized antibodies, antibody-derivedfragments (e.g., single chain antibodies) of any class, find use in thepresent invention. In still other embodiments, structural proteinsincluding but not limited to food allergens (e.g., Ber e 1 [Brazil nutallergen] and Ara H 1 [peanut allergen]) find use in the presentinvention. In additional embodiments, the proteins are industrial and/ormedicinal enzymes. In some embodiments, preferred classes of enzymesinclude, but are not limited to proteases, cellulases, lipases,esterases, amylases, phenol oxidases, oxidases, permeases, pullulanases,isomerases, kinases, phosphatases, lactamases and reductases. As usedherein, “protein” refers to any composition comprised of amino acids andrecognized as a protein by those of skill in the art. The terms“protein,” “peptide” and polypeptide are used interchangeably herein.Amino acids may be referred to by their complete names (e.g., alanine)or by the accepted one letter (e.g., A), or three letter (e.g., ala)abbreviations. Wherein a peptide is a portion of a protein, those skillin the art understand the use of the term in context. The term “protein”encompasses mature forms of proteins, as well as the pro- andprepro-forms of related proteins. Prepro forms of proteins comprise themature form of the protein having a prosequence operably linked to theamino terminus of the protein, and a “pre-” or “signal” sequenceoperably linked to the amino terminus of the prosequence. In preferredembodiments, the protein is a protease. In some particularly preferredembodiments, the protease is a subtilisin, while in alternativeparticularly preferred embodiments, the protease is a subtilisinCarlsberg.

As used herein, “wild-type” and “native” proteins are those found innature. The terms “wild-type sequence,” and “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project.

As used herein, “protease” refers to naturally-occurring proteases, aswell as recombinant proteases. Proteases are carbonyl hydrolases whichgenerally act to cleave peptide bonds of proteins or peptides.Naturally-occurring proteases include, but are not limited to suchexamples as α-aminoacylpeptide hydrolase, peptidylamino acid hydrolase,acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase,thiol proteinase, carboxylproteinase and metalloproteinase. Serine,metallo, thiol and acid proteases are included, as well as endo andexo-proteases. Indeed, in some preferred embodiments, serine proteasessuch as chymotrypsin and subtilisin find use. Both of these serineproteases have a catalytic triad comprising aspartate, histidine andserine. In the subtilisin proteases, the relative order of these aminoacids reading from the carboxy terminus is aspartate-histidine-serine,while in the chymotrypsin proteases, the relative order of these aminoacids reading from the carboxy terminus is histidine-asparate-serine.Although subtilisins are typically obtained from bacterial, fungal oryeast sources, “subtilisin” as used herein, refers to a serine proteasehaving the catalytic triad of the subtilisin proteases defined above.Additionally, human subtilisins are proteins of human origin havingsubtilisin catalytic activity, for example the kexin family of humanderived proteases. Subtilisins are well known by those skilled in theart for example, Bacillus amyloliquefaciens subtilisin (BPN′), Bacilluslentus subtilisin, Bacillus subtilis subtilisin, Bacillus licheniformissubtilisin (See e.g., U.S. Pat. No. 4,760,025 (RE 34,606), U.S. Pat. No.5,204,015, U.S. Pat. No. 5,185,258, European Patent No. 0 328 299, andWO89/06279). In some preferred embodiments of the present invention, theterm is used in reference to a subtilisin, while in particularlypreferred embodiments, the term refers to the ALCALASE® enzyme.

As used herein, “protein” refers to any composition comprised of aminoacids and recognized as a protein by those of skill in the art. Theterms “protein,” “peptide” and polypeptide are used interchangeablyherein. Wherein a peptide is a portion of a protein, those skill in theart understand the use of the term in context. The term “protein”encompasses mature forms of proteins, as well as the pro- andprepro-forms of related proteins. Prepro forms of proteins comprise themature form of the protein having a prosequence operably linked to theamino terminus of the protein, and a “pre-” or “signal” sequenceoperably linked to the amino terminus of the prosequence. As usedherein, functionally similar proteins are considered to be “relatedproteins.” In some embodiments, these proteins are derived from adifferent genus and/or species (e.g., B. subtilis subtilisin and B.lentus subtilisin), including differences between classes of organisms(e.g., a bacterial subtilisin and a fungal subtilisin). In additionalembodiments, related proteins are provided from the same species.Indeed, it is not intended that the present invention be limited torelated proteins from any source(s).

As used herein, the term “derivative” refers to a protein (e.g., aprotease) which is derived from a precursor protein (e.g., the nativeprotease) by addition of one or more amino acids to either or both theC- and N-terminal end(s), substitution of one or more amino acids at oneor a number of different sites in the amino acid sequence, and/ordeletion of one or more amino acids at either or both ends of theprotein or at one or more sites in the amino acid sequence, and/orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of a protease derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative protease.

One type of related (and derivative) proteins are “variant proteins.” Inpreferred embodiments, variant proteins differ from a parent protein andone another by a small number of amino acid residues. The number ofdiffering amino acid residues may be one or more, preferably 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In one preferredembodiment, the number of different amino acids between variants isbetween 1 and 10. In particularly preferred embodiments, relatedproteins and particularly variant proteins comprise at least 50%, 60%,65%. 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequenceidentity. Additionally, a related protein or a variant protein as usedherein, refers to a protein that differs from another related protein ora parent protein in the number of prominent regions. For example, insome embodiments, variant proteins have 1, 2, 3, 4, 5, or 10corresponding prominent regions which differ from the parent protein. Inone embodiment, the prominent corresponding region of a variant producesonly a background level of immunogenic response.

As used herein, “corresponding to,” refers to a residue at theenumerated position in a protein or peptide, or a residue that isanalogous, homologous, or equivalent to an enumerated residue in anotherprotein or peptide.

As used herein, “corresponding region” generally refers to an analogousposition within related proteins or a parent protein.

As used herein, the term “analogous sequence” refers to a sequencewithin a protein that provides similar function, tertiary structure,and/or conserved residues as the protein of interest. In particularlypreferred embodiments, the analogous sequence involves sequence(s) at ornear an epitope. For example, in epitope regions that contain an alphahelix or a beta sheet structure, the replacement amino acids in theanalogous sequence preferably maintain the same specific structure. Theterm also refers to nucleotide sequences, as well as amino acidsequences.

As used herein, “homologous protein” refers to a protein (e.g.,protease) that has similar catalytic action, structure, antigenic,and/or immunogenic response as the protein (i.e., protease) of interest.It is not intended that a homolog and a protein (e.g., protease) ofinterest be necessarily related evolutionarily. Thus, it is intendedthat the term encompass the same functional protein obtained fromdifferent species. In some preferred embodiments, it is desirable toidentify a homolog that has a tertiary and/or primary structure similarto the protein of interest, as replacement for the epitope in theprotein of interest with an analogous segment from the homolog willreduce the disruptiveness of the change. Thus, in most cases, closelyhomologous proteins provide the most desirable sources of epitopesubstitutions. Alternatively, it is advantageous to look to humananalogs for a given protein.

In preferred embodiments, “homolog,” as used herein means an enzymewhich has similar catalytic action, structure and/or use as ALCALASE®enzyme. In some preferred embodiments, the ALCALASE® enzyme homologs ofthe present invention have tertiary and/or primary structuressubstantially similar to wild-type ALCALASE® enzyme. A significantALCALASE® enzyme epitope may be replaced with an analogous segment froma homologous enzyme. This type of replacement may reduce thedisruptiveness of the change in the parent subtilisin. In most cases,closely homologous proteins provide the most desirable source of epitopesubstitutions.

As used herein, “homologous genes” refers to at least a pair of genesfrom different, but usually related species, which correspond to eachother and which are identical or very similar to each other. The termencompasses genes that are separated by speciation (i.e., thedevelopment of new species) (e.g., orthologous genes), as well as genesthat have been separated by genetic duplication (e.g., paralogousgenes).

As used herein, “ortholog” and “orthologous genes” refer to genes indifferent species that have evolved from a common ancestral gene (i.e.,a homologous gene) by speciation. Typically, orthologs retain the samefunction in during the course of evolution. Identification of orthologsfinds use in the reliable prediction of gene function in newly sequencedgenomes.

As used herein, “paralog” and “paralogous genes” refer to genes that arerelated by duplication within a genome. While orthologs retain the samefunction through the course of evolution, paralogs evolve new functions,even though some functions are often related to the original one.Examples of paralogous genes include, but are not limited to genesencoding trypsin, chymotrypsin, elastase, and thrombin, which are allserine proteinases and occur together within the same species.

The degree of homology between sequences may be determined using anysuitable method known in the art (See e.g., Smith and Waterman, Adv.Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443[1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988];programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res., 12:387-395 [1984]).

For example, PILEUP is a useful program to determine sequence homologylevels. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol.,35:351-360 [1987]). The method is similar to that described by Higginsand Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUPparameters including a default gap weight of 3.00, a default gap lengthweight of 0.10, and weighted end gaps. Another example of a usefulalgorithm is the BLAST algorithm, described by Altschul et al.,(Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). One particularly usefulBLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth.Enzymol., 266:460-480 [1996]). parameters “W,” “T,” and “X” determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989])alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

As used herein, “percent (%) nucleic acid sequence identity” is definedas the percentage of nucleotide residues in a candidate sequence thatare identical with the nucleotide residues of the sequence.

As used herein, the term “hybridization” refers to the process by whicha strand of nucleic acid joins with a complementary strand through basepairing, as known in the art.

As used herein, “maximum stringency” refers to the level ofhybridization that typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The phrases “substantially similar and “substantially identical” in thecontext of two nucleic acids or polypeptides typically means that apolynucleotide or polypeptide comprises a sequence that has at least 75%sequence identity, preferably at least 80%, more preferably at least90%, still more preferably 95%, most preferably 97%, sometimes as muchas 98% and 99% sequence identity, compared to the reference (i.e.,wild-type) sequence. Sequence identity may be determined using knownprograms such as BLAST, ALIGN, and CLUSTAL using standard parameters.(See e.g., Altschul, et al., J. Mol. Biol. 215:403-410 [1990]; Henikoffet al., Proc. Natl. Acad. Sci. USA 89:10915 [1989]; Karin et al., Proc.Natl Acad. Sci USA 90:5873 [1993]; and Higgins et al., Gene 73:237-244[1988]). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. Also,databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad.Sci. USA 85:2444-2448 [1988]).

In some embodiments, “equivalent residues” are defined by determininghomology at the level of tertiary structure for a precursor protein(i.e., protein of interest) whose tertiary structure has been determinedby x-ray crystallography. Equivalent residues are defined as those forwhich the atomic coordinates of two or more of the main chain atoms of aparticular amino acid residue of the precursor protein and anotherprotein are within 0.13 nm and preferably 0.1 nm after alignment.Alignment is achieved after the best model has been oriented andpositioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the protein. In most embodiments, the bestmodel is the crystallographic model giving the lowest R factor forexperimental diffraction data at the highest resolution available.

In some embodiments, modification is preferably made to the “precursorDNA sequence” which encodes the amino acid sequence of the precursorenzyme, but can be by the manipulation of the precursor protein. In thecase of residues which are not conserved, the replacement of one or moreamino acids is limited to substitutions which produce a variant whichhas an amino acid sequence that does not correspond to one found innature. In the case of conserved residues, such replacements should notresult in a naturally-occurring sequence. Derivatives provided by thepresent invention further include chemical modification(s) that changethe characteristics of the protease. In some preferred embodiments, theprotein gene is ligated into an appropriate expression plasmid. Thecloned protein gene is then used to transform or transfect a host cellin order to express the protein gene. This plasmid may replicate inhosts in the sense that it contains the well-known elements necessaryfor plasmid replication or the plasmid may be designed to integrate intothe host chromosome. The necessary elements are provided for efficientgene expression (e.g., a promoter operably linked to the gene ofinterest). In some embodiments, these necessary elements are supplied asthe gene's own homologous promoter if it is recognized, (i.e.,transcribed, by the host), a transcription terminator (a polyadenylationregion for eukaryotic host cells) which is exogenous or is supplied bythe endogenous terminator region of the protein gene. In someembodiments, a selection gene such as an antibiotic resistance gene thatenables continuous cultural maintenance of plasmid-infected host cellsby growth in antimicrobial-containing media is also included.

The present invention encompasses proteases having alteredimmunogenicity that are equivalent to those that are derived from theparticular microbial strain mentioned. Being “equivalent,” means thatthe proteases are encoded by a polynucleotide capable of hybridizing tothe polynucleotide having the sequence as shown in any one of thoseshown in FIG. 1, under conditions of medium to high stringency and stillretaining the altered immunogenic response to human T-cells. Being“equivalent” means that the protease comprises at least 55%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97% or at least 99% identity to the epitopesequences and the variant proteases having such epitopes (e.g., havingthe amino acid sequence modified).

As used herein, the terms “hybrid proteases” and “fusion proteases”refer to proteins that are engineered from at least two different or“parental” proteins. In preferred embodiments, these parental proteinsare homologs of one another. For example, in some embodiments, apreferred hybrid protease or fusion protein contains the N-terminus of aprotein and the C-terminus of a homolog of the protein. In somepreferred embodiment, the two terminal ends are combined to correspondto the full-length active protein. In alternative preferred embodiments,the homologs share substantial similarity but do not have identicalT-cell epitopes. Therefore, in one embodiment, the present inventionprovides a protease of interest having one or more T-cell epitopes inthe C-terminus, but in which the C-terminus is replaced with theC-terminus of a homolog having a less potent T-cell epitope, or fewer orno T-cell epitopes in the C-terminus. Thus, the skilled artisanunderstands that by being able to identify T-cell epitopes amonghomologs, a variety of variants producing different immunogenicresponses can be formed. Moreover, it is understood that internalportions, and more than one homolog can be used to produce the variantsof the present invention.

The variants of the present invention include the mature forms ofprotein variants, as well as the pro- and prepro-forms of such proteinvariants. The prepro-forms are the preferred construction since thisfacilitates the expression, secretion and maturation of the proteinvariants.

As used herein, “prosequence” refers to a sequence of amino acids boundto the N-terminal portion of the mature form of a protein which whenremoved results in the appearance of the “mature” form of the protein.Many proteolytic enzymes are found in nature as translational proenzymeproducts and, in the absence of post-translational processing, areexpressed in this fashion. A preferred prosequence for producing proteinvariants such as protease variants is the putative prosequence ofBacillus licheniformis subtilisin Carlsberg, although other prosequencesfind use in the present invention.

As used herein, “signal sequence” and “presequence” refer to anysequence of amino acids bound to the N-terminal portion of a protein orto the N-terminal portion of a pro-protein which may participate in thesecretion of the mature or pro forms of the protein. This definition ofsignal sequence is a functional one and is intended to include all thoseamino acid sequences encoded by the N-terminal portion of the proteingene which participate in the effectuation of the secretion of proteinunder native conditions. The present invention utilizes such sequencesto effect the secretion of the protein variants described herein.

As used herein, a “prepro” form of a protein variant consists of themature form of the protein having a prosequence operably linked to theamino terminus of the protein and a “pre” or “signal” sequence operablylinked to the amino terminus of the prosequence.

The term “regulatory element” as used herein refers to a genetic elementthat controls some aspect of the expression of nucleic acid sequences.For example, a promoter is a regulatory element which facilitates theinitiation of transcription of an operably linked coding region.Additional regulatory elements include splicing signals, polyadenylationsignals and termination signals.

As used herein, “expression vector” refers to a DNA construct containinga DNA sequence that is operably linked to a suitable control sequencecapable of effecting the expression of the DNA in a suitable host. Suchcontrol sequences include a promoter to effect transcription, anoptional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid” and“vector” are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors that serveequivalent functions and which are, or become, known in the art.

As used herein, “host cells” are generally prokaryotic or eukaryotichosts that preferably have been manipulated by the methods known in theart (See e.g., U.S. Pat. No. 4,760,025 (RE 34,606)) to render themincapable of secreting enzymatically active endoprotease. A preferredhost cell for expressing protein is the Bacillus strain BG2036 which isdeficient in enzymatically active neutral protein and alkaline protease(subtilisin). The construction of strain BG2036 is described in detailin U.S. Pat. No. 5,264,366. Other host cells for expressing proteininclude Bacillus subtilis 1168 (also described in U.S. Pat. No.4,760,025 (RE 34,606) and U.S. Pat. No. 5,264,366), as well as anysuitable Bacillus strain, including those within the species of B.licheniformis, B. lentus, and other Bacillus species, etc.

Host cells are transformed or transfected with vectors constructed usingrecombinant DNA techniques known in the art. Transformed host cells arecapable of either replicating vectors encoding the protein variants orexpressing the desired protein variant. In the case of vectors whichencode the pre- or prepro-form of the protein variant, such variants,when expressed, are typically secreted from the host cell into the hostcell medium.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means transformation, transduction ortransfection. Means of transformation include protoplast transformation,calcium chloride precipitation, electroporation, naked DNA and the likeas known in the art. (See, Chang and Cohen (1979) Mol. Gen. Genet.168:111-115; Smith et al., (1986) Appl. and Env. Microbiol. 51:634; andthe review article by Ferrari et al., (1989) Genetics, pages 57-72 inBacillus ed. C. Harwood, Plenum Publishing Corporation).

In embodiments involving proteases, variant protease activity can bedetermined and compared with the protease of interest by examining theinteraction of the protease with various commercial substrates,including, but not limited to casein, keratin, elastin, and collagen.Indeed, protease activity can be determined by any suitable method knownin the art. Exemplary assays to determine protease activity include, butare not limited to, succinyl-Ala-Ala-Pro-Phe-para nitroanilide(SAAPFpNA) (citation) assay; and 2,4,6-trinitrobenzene sulfonate sodiumsalt (TNBS) assay. In the SAAPFpNA assay, proteases cleave the bondbetween the peptide and p-nitroaniline to give a visible yellow colourabsorbing at 405 nm. In the TNBS color reaction method, the assaymeasures the enzymatic hydrolysis of the substrate into polypeptidescontaining free amino groups. These amino groups react with TNBS to forma yellow colored complex. Thus, the more deeply colored the reaction,the more activity is measured. The yellow color can be determined byvarious analyzers or spectrophotometers known in the art.

Other characteristics of the variant proteases can be determined bymethods known to those skilled in the art. Exemplary characteristicsinclude, but are not limited to thermal stability, alkaline stability,and stability of the particular protease in various substrate or buffersolutions or product formulations.

When combined with the enzyme stability assay procedures disclosedherein, mutants obtained by random mutagenesis can be identified whichdemonstrate either increased or decreased alkaline or thermal stabilitywhile maintaining enzymatic activity.

Alkaline stability can be measured either by known procedures or by themethods described herein. A substantial change in alkaline stability isevidenced by at least about a 5% or greater increase or decrease (inmost embodiments, it is preferably an increase) in the half-life of theenzymatic activity of a mutant when compared to the precursor protein.

Thermal stability can be measured either by known procedures or by themethods described herein. A substantial change in thermal stability isevidenced by at least about a 5% or greater increase or decrease (inmost embodiments, it is preferably an increase) in the half-life of thecatalytic activity of a mutant when exposed to a relatively hightemperature and neutral pH as compared to the precursor protein.

As used herein, “personal care products” means products used in thecleaning of hair, skin, scalp, teeth, including, but not limited toshampoos, body lotions, shower gels, topical moisturizers, toothpaste,and/or other topical cleansers. In some particularly preferredembodiments, these products are utilized by humans, while in otherembodiments, these products find use with non-human animals (e.g., inveterinary applications).

As used herein, “skin care compositions” means products used in topicalapplication for cleaning and/or moisturizing skin. Such compositionsinclude, but are not limited to moisturizing body washes, shower gels,body lotions, moisturizing facial creams, make-up removers, and lotions.

As used herein, “cleaning compositions” are compositions that can beused to remove undesired compounds from substrates, such as fabric,dishes, contact lenses, other solid substrates, hair (shampoos), skin(soaps and creams), teeth (mouthwashes, toothpastes) etc.

The term “cleaning composition materials,” as used herein, refers to anyliquid, solid or gaseous material selected for the particular type ofcleaning composition desired and the form of the product (e.g., liquid;granule; spray composition), which materials are also compatible withthe protease enzyme used in the composition. The specific selection ofcleaning composition materials are readily made by considering thesurface, item or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use (e.g., through thewash detergent use).

As used herein the term “hard surface cleaning composition,” refers todetergent compositions for cleaning hard surfaces such as floors, walls,bathroom tile, and the like. Such compositions are provided in any form,including but not limited to solids, liquids, emulsions, etc.

As used herein, “dishwashing composition” refers to all forms forcompositions for cleaning dishes, including but not limited to, granularand liquid forms.

As used herein, “fabric cleaning composition” refers to all forms ofdetergent compositions for cleaning fabrics, including but not limitedto, granular, liquid and bar forms. As used herein, “fabric” refers toany textile material.

As used herein, the term “compatible,” means that the cleaningcomposition materials do not reduce the proteolytic activity of theprotease enzyme to such an extent that the protease is not effective asdesired during normal use situations. Specific cleaning compositionmaterials are exemplified in detail hereinafter.

As used herein, “effective amount of protease enzyme” refers to thequantity of protease enzyme (e.g., subtilisin Carlsberg) necessary toachieve the enzymatic activity necessary in the specific application(e.g., personal care product, cleaning composition, etc.). Sucheffective amounts are readily ascertained by one of ordinary skill inthe art and are based on many factors, such as the particular enzymeused, the cleaning application, the specific composition of the cleaningcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, and the like.

As used herein, “non-fabric cleaning compositions” encompass hardsurface cleaning compositions, dishwashing compositions, oral cleaningcompositions, denture cleaning compositions, and personal cleansingcompositions.

As used herein, “oral cleaning compositions” refers to dentifrices,toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouthgels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxispastes, dental treatment solutions, and the like.

As used herein, “pharmaceutically acceptable” means that drugs,medicaments and/or inert ingredients which the term describes aresuitable for use in contact with the tissues of humans and other animalswithout undue toxicity, incompatibility, instability, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio.

Many of the protein variants of the present invention are useful informulating various detergent compositions. A number of known compoundsare suitable surfactants useful in compositions comprising the proteinmutants of the invention. These include nonionic, anionic, cationic,anionic or zwitterionic detergents (See e.g., U.S. Pat. No. 4,404,128and U.S. Pat. No. 4,261,868). A suitable detergent formulation is thatdescribed in U.S. Pat. No. 5,204,015. Those in the art are familiar withthe different formulations which find use as cleaning compositions. Inaddition to typical cleaning compositions, it is readily understood thatthe protein variants of the present invention find use in any purposethat native or wild-type proteins are used. Thus, these variants can beused, for example, in bar or liquid soap applications, dishcareformulations, surface cleaning applications, contact lens cleaningsolutions or products, peptide hydrolysis, waste treatment, textileapplications, as fusion-cleavage enzymes in protein production, etc.Indeed, it is not intended that the variants of the present invention belimited to any particular use. For example, the variants of the presentinvention may comprise, in addition to decreasedallergenicity/immunogenicity, enhanced performance in a detergentcomposition (as compared to the precursor). As used herein, enhancedperformance in a detergent is defined as increasing cleaning of certainenzyme sensitive stains (e.g., grass or blood), as determined by usualevaluation after a standard wash cycle.

Proteins, particularly proteases of the invention can be formulated intoknown powdered and liquid detergents having pH between 6.5 and 12.0 atlevels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. Insome embodiments, these detergent cleaning compositions further includeother enzymes such as proteases, amylases, cellulases, lipases orendoglycosidases, as well as builders and stabilizers.

The addition of proteins to conventional cleaning compositions does notcreate any special use limitations. In other words, any temperature andpH suitable for the detergent are also suitable for the presentcompositions, as long as the pH is within the above range, and thetemperature is below the described protein's denaturing temperature. Inaddition, proteins of the invention find use in cleaning compositionswithout detergents, again either alone or in combination with buildersand stabilizers.

In one embodiment, the present invention provides compositions for thetreatment of textiles that includes variant proteins of the presentinvention. The composition can be used to treat for example silk or wool(See e.g., U.S. Reissue Pat. No. 216,034; European Patent No. 134,267;U.S. Pat. No. 4,533,359; and European Patent No. 344,259). Thesevariants can be screened for proteolytic activity and their suitabilityfor these applications using methods well known in the art.

As indicated above, the proteins of the present invention exhibitmodified immunogenic responses (e.g., antigenicity and/orimmunogenicity) when compared to the native proteins encoded by theirprecursor DNAs. In some preferred embodiments, the proteins (e.g.,proteases) exhibit reduced allergenicity/immunogenicity. Those of skillin the art readily recognize that the uses of the proteases of thisinvention will be determined, in large part, on the immunologicalproperties of the proteins. For example, proteases that exhibit reducedimmunogenic responses can be used in cleaning compositions. An effectiveamount of one or more protease variants described herein find use incompositions useful for cleaning a variety of surfaces in need ofproteinaceous stain removal. Such cleaning compositions includedetergent compositions for cleaning hard surfaces, detergentcompositions for cleaning fabrics, dishwashing compositions, oralcleaning compositions, and denture cleaning compositions.

An effective amount of one or more related and/or variant proteins withreduced allergenicity/immunogenicity, ranked according to the methods ofthe present invention find use in various compositions that are appliedto keratinous materials such as nails and hair, including but notlimited to those useful as hair spray compositions, hair shampoo and/orconditioning compositions, compositions applied for the purpose of hairgrowth regulation, and compositions applied to the hair and scalp forthe purpose of treating seborrhea, dermatitis, and/or dandruff.

An effective amount of one or more protease variant(s) described hereinfind use in included in compositions suitable for topical application tothe skin or hair. These compositions can be in the form of creams,lotions, gels, and the like, and may be formulated as aqueouscompositions or may be formulated as emulsions of one or more oil phasesin an aqueous continuous phase.

In addition, the related and/or variant proteins with reducedallergenicity/immunogenicity find use in other applications, includingpharmaceutical applications, drug delivery applications, and otherhealth care applications.

Skin Care Active

In some embodiments, the compositions provided by the present inventioncomprise a skin care active at a level from about 0.1% to about 20%,preferably from about 1% to about 10%, more preferably from about 2% toabout 8%, by weight. Non-limiting examples of suitable skin care activesfor use herein include a vitamin B₃ component, panthenol, vitamin E,vitamin E acetate, retinol, retinyl propionate, retinyl palmitate,retinoic acid, vitamin C, theobromine, α-hydroxyacid, farnesol,phytantriol, salicylic acid, palmityl peptapeptide-3 and mixturesthereof.

B3 Compound

As used herein, “vitamin B₃ compound” means a compound having theformula:

wherein R is —CONH₂ (i.e., niacinamide), —COOH (i.e., nicotinic acid) or—CH₂OH (i.e., nicotinyl alcohol); derivatives thereof; and salts of anyof the foregoing. Exemplary derivatives of the foregoing vitamin B₃compounds include nicotinic acid esters, including non-vasodilatingesters of nicotinic acid, nicotinyl amino acids, nicotinyl alcoholesters of carboxylic acids, nicotinic acid N-oxide and niacinamideN-oxide.

Suitable esters of nicotinic acid include nicotinic acid esters ofC₁-C₂₂, preferably C₁-C₁₆, more preferably C₁-C₆ alcohols. The alcoholsare suitably straight-chain or branched chain, cyclic or acyclic,saturated or unsaturated (including aromatic), and substituted orunsubstituted. The esters are preferably non-vasodilating. As usedherein, “non-vasodilating” means that the ester does not commonly yielda visible flushing response after application to the skin in the subjectcompositions (i.e., the majority of the general population would notexperience a visible flushing response, although such compounds maycause vasodilation not visible to the naked eye). Non-vasodilatingesters of nicotinic acid include tocopherol nicotinate and inositolhexanicotinate; tocopherol nicotinate is preferred. A more completedescription of vitamin B₃ compounds is given in WO 98/22085. Preferredvitamin B₃ compounds are niacinamide and tocopherol nicotinate.

Retinoids

Another suitable skin care active is a retinoid. As used herein,“retinoid” includes all natural and/or synthetic analogs of Vitamin A orretinol-like compounds which possess the biological activity of VitaminA in the skin as well as the geometric isomers and stereoisomers ofthese compounds. When a retinoid is included in the compositions herein,it typically comprises from or about 0.005% to or about 2%, morepreferably 0.01% to about 2% retinoid. Retinol is preferably used in anamount of from or about 0.01% to or about 0.15%; retinol esters arepreferably used in an amount of from about 0.01% to about 2% (e.g.,about 1%).

The retinoid is preferably retinol, retinol esters (e.g., C₂-C₂₂ alkylesters of retinol, including retinyl palmitate, retinyl acetate, retinylpropionate), retinal, and/or retinoic acid (including all-trans retinoicacid and/or 13-cis-retinoic acid), more preferably retinoids other thanretinoic acid. These compounds are well known in the art and arecommercially available from a number of sources (e.g., Sigma ChemicalCompany (St. Louis, Mo.), and Boehringer Mannheim (Indianapolis, Ind.)).Preferred retinoids include retinol, retinyl palmitate, retinyl acetate,retinyl propionate, retinal, retinoic acid and combinations thereof.More preferred retinoids include retinol, retinoic propionate, retinoicacid and retinyl palmitate. The retinoid may be included as thesubstantially pure material, or as an extract obtained by suitablephysical and/or chemical isolation from natural (e.g., plant) sources.

Carriers

It is further contemplated that the compositions of the presentinvention will find use in safe and effective amounts of adermatologically acceptable carrier, suitable for topical application tothe skin and/or hair within which the essential materials and optionalother materials are incorporated to enable the essential materials andoptional components to be delivered to the skin or hair at anappropriate concentration. Thus, the carrier acts as a diluent,dispersant, solvent, or the like for the essential components whichensures that they can be applied to and distributed evenly over theselected target at an appropriate concentration.

The type of carrier utilized in the present invention depends on thetype of product form desired for the composition. It is not intendedthat the present invention be limited to a carrier of any particularform, although it is most commonly a solid, semi-solid or liquid.Suitable carriers are liquid or semi-solid, such as creams, lotions,gels, sticks, ointments, pastes and mousses. Preferably the carrier isin the form of a lotion, cream or a gel, more preferably one which has asufficient thickness or yield point to prevent the particles fromsedimenting. The carrier can itself be inert or it can possessdermatological benefits of its own. The carrier may be applied directlyto the skin and/or hair, or it may be applied via a woven or non-wovenwipe or cloth. It may also be in the form of a patch, mask, or wrap. Itmay also be aerosolized or otherwise sprayed onto the skin and/or hair.The carrier should also be physically and chemically compatible with theessential components described herein, and should not unduly impairstability, efficacy or other use benefits associated with thecompositions of the present invention.

Preferred carriers contain a dermatologically acceptable, hydrophilicdiluent. Suitable hydrophilic diluents include water, organichydrophilic diluents such as C₁-C₄ monohydric alcohols and low molecularweight glycols and polyols, including propylene is glycol, polyethyleneglycol (e.g. of MW 200-600), polypropylene glycol (e.g. of MW 425-2025),glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters,1,2,6-hexametriol, ethanol, iso-propanol, sorbitol esters, ethoxylatedethers, propoxylated ethers and combinations thereof. The diluent ispreferably liquid. Water is a preferred diluent. The compositionpreferably comprises at least about 20% of the hydrophilic diluent.Suitable carriers may also comprise an emulsion comprising a hydrophilicphase, especially an aqueous phase, and a hydrophobic phase (e.g., alipid, oil or oily material). As well known to those skilled in the art,the hydrophilic phase is dispersed in the hydrophobic phase, or viceversa, to form respectively hydrophilic or hydrophobic dispersed andcontinuous phases, depending on the composition ingredients. In emulsiontechnology, the well-known term “dispersed phase” means that the phaseexists as small particles or droplets that are suspended in andsurrounded by a continuous phase. The dispersed phase is also known asthe internal or discontinuous phase. The emulsion may be or comprise(e.g., in a triple or other multi-phase emulsion) an oil-in-wateremulsion or a water-in-oil emulsion such as a water-in-siliconeemulsion. Oil-in-water emulsions typically comprise from about 1% toabout 60% (preferably about 1% to about 30%) of the dispersedhydrophobic phase and from about 1% to about 99% (preferably from about40% to about 90%) of the continuous hydrophilic phase; water-in-oilemulsions typically comprise from about 1% to about 98% (preferably fromabout 40% to about 90%) of the dispersed hydrophilic phase and fromabout 1% to about 50% (preferably about 1% to about 30%) of thecontinuous hydrophobic phase.

Humectants

In some embodiments, the compositions of the present invention comprisehumectants which are preferably present at a level of from about 0.01%to about 20%, more preferably from about 0.1% to about 15% andespecially from about 0.5% to about 10%. Preferred humectants include,but are not limited to, compounds selected from polyhydric alcohols,urea, D or DL panthenol, calcium pantothenate, royal jelly, panthetine,pantotheine, panthenyl ethyl ether, pangamic acid, pyridoxin, pantoyllactose Vitamin B complex, hexane-1,2,6,-triol, guanidine or itsderivatives, and mixtures thereof.

Suitable polyhydric alcohols for use herein include polyalkylene glycolsand more preferably alkylene polyols and their derivatives, includingpropylene glycol, dipropylene glycol, polypropylene glycol, polyethyleneglycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol,erythritol, threitol, pentaerythritol, xylitol, glucitol, mannitol,hexylene glycol, butylene glycol (e.g., 1,3-butylene glycol), hexanetriol (e.g., 1,2,6-hexanetriol), trimethylol propane, neopentyl glycol,glycerine, ethoxylated glycerine, propane-1,3 diol, propoxylatedglycerine and mixtures thereof. The alkoxylated derivatives of any ofthe above polyhydric alcohols are also suitable for use herein.Preferred polyhydric alcohols of the present invention are selected fromglycerine, butylene glycol, propylene glycol, dipropylene glycol,polyethylene glycol, hexane triol, ethoxylated glycerine andpropoxylated glycerine, and mixtures thereof.

Suitable humectants useful herein are sodium 2-pyrrolidone-5-carboxylate(NaPCA), guanidine; glycolic acid and glycolate salts (e.g. ammonium andquaternary alkyl ammonium); lactic acid and lactate salts (e.g. ammoniumand quaternary alkyl ammonium); aloe vera in any of its variety of forms(e.g., aloe vera gel); hyaluronic acid and derivatives thereof (e.g.,salt derivatives such as sodium hyaluronate); lactamidemonoethanolamine; acetamide monoethanolamine; urea; panthenol andderivatives thereof; and mixtures thereof.

At least part (up to about 5% by weight of composition) of a humectantcan be incorporated in the form of an admixture with a particulatecross-linked hydrophobic acrylate or methacrylate copolymer, itselfpreferably present in an amount of from about 0.1% to about 10%, whichcan be added either to the aqueous or disperse phase. This copolymer isparticularly valuable for reducing shine and controlling oil whilehelping to provide effective moisturization benefits and is described infurther detail by WO96/03964, incorporated herein by reference.

Emollients

In some embodiments, the oil in water emulsion embodiments of thepresent invention comprise from about 1% to about 20%, preferably fromabout 1.5% to about 15%, more preferably from about 0.1% to about 8%,and even more preferably from about 0.5% to about 5% of adermatologically acceptable emollient. Emollients tend to lubricate theskin, increase the smoothness and suppleness, prevent or relievedryness, and/or protect the skin. Emollients are typicallywater-immiscible, oily or waxy materials and emollients with highmolecular weights can confer tacky properties to a topical composition.A wide variety of suitable emollients are known and may be used herein.For example, Sagarin, Cosmetics, Science and Technology, 2nd Edition,Vol. 1, pp. 3243 (1972), contains numerous examples of materialssuitable for use as emollients. In addition, all emollients discussed inapplication WO 00/24372 should be considered as suitable for use in thepresent invention although preferred examples are outlined in furtherdetail below:

-   -   i) Straight and branched chain hydrocarbons having from about 7        to about 40 carbon atoms, such as dodecane, squalane,        cholesterol, hydrogenated polyisobutylene, isohexadecane,        isoeicosane, isooctahexacontane, isohexapentacontahectane, and        the C₇-C₄₀ isoparaffins, which are C₇-C₄₀ branched hydrocarbons.        Suitable branched chain hydrocarbons for use herein are selected        from isopentacontaoctactane, petrolatum, and mixtures thereof.        Suitable for use herein are branched chain aliphatic        hydrocarbons sold under the trade name Permethyl (RTM) and        commercially available from Presperse Inc., South Plainfield,        N.J.    -   ii) C₁-C₃₀ alcohol esters of C₁-C₃₀ carboxylic acids, C12-15        alkyl benzoates, and of C₂-C₃₀ dicarboxylic acids, for example,        isononyl isononanoate, isostearyl neopentanoate. isodecyl        octanoate, isodecyl isononanoate, tridecyl isononanoate,        myristyl octanoate, octyl pelargonate, octyl isononanoate,        myristyl myristate, myristyl neopentanoate, myristyl octanoate,        isopropyl myristate, myristyl propionate, isopropyl stearate,        isopropyl isostearate, methyl isostearate, behenyl behenate,        dioctyl maleate, diisopropyl adipate, and diisopropyl        dilinoleate and mixtures thereof.    -   iii) C₁-C₃₀ mono- and poly-esters of sugars and related        materials. These esters are derived from a sugar or polyol        moiety and one or more carboxylic acid moieties. Depending on        the constituent acid and sugar, these esters can be in either        liquid or solid form at room temperature. Examples include        glucose tetraoleate, the galactose tetraesters of oleic acid,        the sorbitol tetraoleate, sucrose tetraoleate, sucrose        pentaoleate, sucrose hexaoleate, sucrose heptaoleate, sucrose        octaoleate, sorbitol hexaester in which the carboxylic acid        ester moieties are palmitoleate and arachidate in a 1:2 molar        ratio, and the octaester of sucrose wherein the esterifying        carboxylic acid moieties are laurate, linoleate and behenate in        a 1:3:4 molar ratio. Other materials include cottonseed oil or        soybean oil fatty acid esters of sucrose. Other examples of such        materials are described in WO 96/16636. A particularly preferred        material is known by the INCI name sucrose polycottonseedate.    -   iv) Vegetable oils and hydrogenated vegetable oils. Examples of        vegetable oils and hydrogenated vegetable oils include safflower        oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil,        palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil,        rice bran oil, pine oil, sesame oil, sunflower seed oil,        partially and fully hydrogenated oils from the foregoing        sources, and mixtures thereof.    -   v) Soluble or colloidally-soluble moisturizing agents. Examples        include hylaronic acid and starch-grafted sodium polyacrylates        such as Sanwet (RTM) IM-1000, IM-1500 and IM-2500 available from        Celanese Superabsorbent Materials, Portsmith, Va., and described        in U.S. Pat. No. 4,076,663.

Preferred emollients for use herein are isohexadecane, isooctacontane,petrolatum, isononyl isononanoate, isodecyl octanoate, isodecylisononanoate, tridecyl isononanoate, myristyl octanoate, octylisononanoate, myristyl myristate, methyl isostearate, isopropylisostearate, C12-15 alkyl benzoates and mixtures thereof. Particularlypreferred emollients for use herein are isohexadecane, isononylisononanoate, methyl isostearate, isopropyl isostearate, petrolatum, ormixtures thereof.

Emulsifiers/Surfactants

In some embodiments, the compositions of the present invention containan emulsifier and/or surfactant, generally to help disperse and suspendthe disperse phase within the continuous aqueous phase. A surfactant mayalso be useful if the product is intended for skin cleansing. Forconvenience hereinafter, emulsifiers are encompassed within the term“surfactants.” Thus, “surfactant(s)” refer(s) to surface active agentswhether used as emulsifiers or for other surfactant purposes such asskin cleansing. Known or conventional surfactants find use used in thecompositions of the present invention, provided that the selected agentis chemically and physically compatible with essential components of thecomposition, and provides the desired characteristics. Suitablesurfactants include non-silicone derived materials, and mixturesthereof. All surfactants discussed in application WO 00/24372 areconsidered as suitable for use in the present invention.

In some embodiments, the compositions of the present invention comprisefrom about 0.05% to about 15% of a surfactant or mixture of surfactants.The exact surfactant or surfactant mixture chosen will depend upon thepH of the composition and the other components present.

Among the nonionic surfactants that are useful herein are those that canbe broadly defined as condensation products of long chain alcohols (e.g.C₈₋₃₀ alcohols), with sugar or starch polymers (i.e., glycosides). Otheruseful nonionic surfactants include the condensation products ofalkylene oxides with fatty acids (i.e., alkylene oxide esters of fattyacids). These materials have the general formula RCO(X)_(n)OH wherein Ris a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e., derived from ethyleneglycol or oxide) or —OCH₂CHCH₃— (i.e., derived from propylene glycol oroxide), and n is an integer from about 6 to about 200. Other nonionicsurfactants are the condensation products of alkylene oxides with 2moles of fatty acids (i.e., alkylene oxide diesters of fatty acids).These materials have the general formula RCO(X)_(n)OOCR wherein R is aC₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e. derived from ethylene glycol oroxide) or —OCH₂CHCH₃— (i.e., derived from propylene glycol or oxide),and n is an integer from about 6 to about 100. An emulsifier for useherein is most preferably a fatty acid ester blend based on a mixture ofsorbitan fatty acid ester and sucrose fatty acid ester, especially ablend of sorbiton stearate and sucrose cocoate. This is commerciallyavailable from ICI under the trade name Arlatone 2121. Even furthersuitable examples include a mixture of cetearyl alcohols, cetearylglucosides such as those available under the trade name Montanov 68 fromSeppic and Emulgade PL68/50 available from Henkel.

In some embodiments, the hydrophilic surfactants useful hereinalternatively or additionally include any of a wide variety of cationic,anionic, zwitterionic, and amphoteric surfactants such as are known inthe art (See e.g., U.S. Pat. No. 5,011,681, U.S. Pat. No. 4,421,769, andU.S. Pat. No. 3,755,560). A wide variety of anionic surfactants alsofind use in the compositions of the present invention (See e.g., U.S.Pat. No. 3,929,678). Exemplary anionic surfactants include the alkoylisethionates (e.g., C₁₂-C₃₀), alkyl and alkyl ether sulfates and saltsthereof, alkyl and alkyl ether phosphates and salts thereof, alkylmethyl taurates (e.g., C₁₂-C₃₀), and soaps (e.g., alkali metal salts,such as sodium or potassium salts) of fatty acids.

Amphoteric and zwitterionic surfactants also find use in thecompositions of the present invention. Examples of amphoteric andzwitterionic surfactants which can be used in the compositions of thepresent invention are those which are broadly described as derivativesof aliphatic secondary and tertiary amines in which the aliphaticradical can be straight or branched chain and wherein one of thealiphatic substituents contains from about 8 to about 22 carbon atoms(preferably C₈-C₁₈) and one contains an anionic water solubilizing group(e.g., carboxy, sulfonate, sulfate, phosphate, or phosphonate). Examplesinclude alkyl imino acetates, iminodialkanoates and aminoalkanoates,imidazolinium and ammonium derivatives. Other suitable amphoteric andzwitterionic surfactants include those selected from the groupconsisting of betaines, sultaines, hydroxysultaines, and branched andunbranched alkanoyl sarcosinates, and mixtures thereof.

In some embodiments, emulsions of the present invention further includea silicone containing emulsifier or surfactant. A wide variety ofsilicone emulsifiers find use in the present invention. These siliconeemulsifiers are typically organically modified organopolysiloxanes, alsoknown to those skilled in the art as silicone surfactants. Usefulsilicone emulsifiers include dimethicone copolyols. These materials arepolydimethyl siloxanes which have been modified to include polyetherside chains such as polyethylene oxide chains, polypropylene oxidechains, mixtures of these chains, and polyether chains containingmoieties derived from both ethylene oxide and propylene oxide. Otherexamples include alkyl-modified dimethicone copolyols (i.e., compoundswhich contain C₂-C₃₀ pendant side chains). Still other usefuldimethicone copolyols include materials having various cationic,anionic, amphoteric, and zwitterionic pendant moieties.

Polymeric Thickening Agents

In some embodiments, the compositions of the present invention compriseat least one polymeric thickening agent. The polymeric thickening agentsuseful herein preferably have a number average molecular weight ofgreater than 20,000, more preferably greater than 50,000 and especiallygreater than 100,000. In some embodiments, the compositions of thepresent invention comprise from about 0.01% to about 10%, preferablyfrom about 0.1% to about 8% and most preferably from about 0.5% to about5% by weight of the composition of the polymeric thickening agent, ormixtures thereof.

Preferred polymer thickening agents for use herein include non-ionicthickening is agents and anionic thickening agents, or mixtures thereof.Suitable non-ionic thickening agents include polyacrylamide polymers,crosslinked poly(N-vinylpyrrolidones), polysaccharides, natural orsynthetic gums, polyvinylpyrrolidone, and polyvinylalcohol. Suitableanionic thickening agents include acrylic acid/ethyl acrylatecopolymers, carboxyvinyl polymers and crosslinked copolymers of alkylvinyl ethers and maleic anhydride. Particularly preferred thickeningagents for use herein are the non-ionic polyacrylamide polymers such aspolyacrylamide and isoparaffin and laureth-7, available under the tradename Sepigel 305 from Seppic Corporation, and acrylic acid/ethylacrylate copolymers and the carboxyvinyl polymers sold by the B.F.Goodrich Company under the trade mark of CARBOPOL™ resins, or mixturesthereof. In some embodiments, suitable CARBOPOL™ resins arehydrophobically modified. Additional suitable resins are described inWO98/22085. It is also contemplated that mixtures of these resins willfind use in the present invention.

Silicone Oil

In some embodiments, the present compositions comprise, at least onesilicone oil phase. Silicone oil phase(s) generally comprises from about0.1% to about 20%, preferably from about 0.5% to about 10%, morepreferably from about 0.5% to about 5%, of the composition. The, oreach, silicone oil phase preferably comprises one or more siliconecomponents.

In some embodiments, silicone components are fluids, including straightchain, branched and cyclic silicones. Suitable silicone fluids usefulherein include silicones inclusive of polyalkyl siloxane fluids,polyaryl siloxane fluids, cyclic and linear polyalkylsiloxanes,polyalkoxylated silicones, amino and quaternary ammonium modifiedsilicones, polyalkylaryl siloxanes or a polyether siloxane copolymer andmixtures thereof. The silicone fluids can be volatile or non-volatile.Silicone fluids generally have a weight average molecular weight of lessthan about 200,000. Suitable silicone fluids have a molecular weight ofabout 100,000 or less, preferably about 50,000 or less, most preferablyabout 10,000 or less. Preferably the silicone fluid is selected fromsilicone fluids having a weight average molecular weight in the rangefrom about 100 to about 50,000 and preferably from about 200 to about40,000. Typically, silicone fluids have a viscosity ranging from about0.65 to about 600,000 mm².s⁻¹, preferably from about 0.65 to about10,000 mm².s⁻¹ at 25° C. The viscosity can be measured by means of aglass capillary viscometer as set forth in Dow Corning Corporate TestMethod CTM0004. Suitable polydimethyl siloxanes that find use in thepresent invention include those available, for example, from the GeneralElectric Company as the SF and Viscasil (RTM) series and from DowCorning as the Dow Corning 200 series. Also useful are essentiallynon-volatile polyalkylarylsiloxanes (e.g., polymethylphenylsiloxanes),having viscosities of about 0.65 to 30,000 mm².s⁻¹ at 25° C. Thesesiloxanes are available, for example, from the General Electric Companyas SF 1075 methyl phenyl fluid or from Dow Corning as 556 Cosmetic GradeFluid. Cyclic polydimethylsiloxanes suitable for use herein are thosehaving a ring structure incorporating from about 3 to about 7 (CH₃)₂SiOmoieties.

Silicone gums also find use with the present invention. The term“silicone gum” herein means high molecular weight silicones having aweight average molecular weight in excess of about 200,000 andpreferably from about 200,000 to about 4,000,000. The present inventionincludes non-volatile polyalkyl as well as polyaryl siloxane gums. Inpreferred embodiments, a silicone oil phase comprises a silicone gum ora mixture of silicones including the silicone gum. Typically, siliconegums have a viscosity at 25° C. in excess of about 1,000,000 mm².s⁻¹.The silicone gums include dimethicones as known in the art (See e.g.,U.S. Pat. No. 4,152,416), as well as the silicone gums described inGeneral Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54and SE 76. Specific examples of silicone gums includepolydimethylsiloxane, (polydimethylsiloxane)(methylvinylsiloxane)copolymer, poly(dimethylsiloxane)(diphenyl)(methylvinylsiloxane)copolymer and mixtures thereof. Preferred silicone gums for use hereinare silicone gums having a molecular weight of from about 200,000 toabout 4,000,000 selected from dimethiconol, dimethicone copolyol,dimethicone, and mixtures thereof.

A silicone phase herein preferably comprises a silicone gum incorporatedinto the composition as part of a silicone gum-fluid blend. When thesilicone gum is incorporated as part of a silicone gum-fluid blend, thesilicone gum preferably constitutes from about 5% to about 40%,especially from about 10% to 20% by weight of the silicone gum-fluidblend. Suitable silicone gum-fluid blends herein are mixtures consistingessentially of:

-   (i) a silicone having a molecular weight of from about 200,000 to    about 4,000,000 selected from dimethiconol, fluorosilicone and    dimethicone and mixtures thereof; and-   (ii) a carrier which is a silicone fluid, the carrier having a    viscosity from about 0.65 mm².s⁻¹ to about 100 mm².s⁻¹,    wherein the ratio of i) to ii) is from about 10:90 to about 20:80    and wherein the silicone gum-based component has a final viscosity    of from about 100 mm².s⁻¹ to about 100,000 is mm².s⁻¹, preferably    from 500 mm².s⁻¹ to about 10,000 mm².s^(−1.)

Further silicone components suitable for use in a silicone oil phaseherein are crosslinked polyorganosiloxane polymers, optionally dispersedin a fluid carrier. In general, crosslinked polyorganosiloxane polymers,together with its carrier (if present) comprise 0.1% to about 20%,preferably from about 0.5% to about 10%, more preferably from about 0.5%to about 5% of the composition. Such polymers comprisepolyorganosiloxane polymers crosslinked by a crosslinking agent.Suitable crosslinking agents include those described in WO98/22085.Examples of suitable polyorganosiloxane polymers for use herein includemethyl vinyl dimethicone, methyl vinyl diphenyl dimethicone, and methylvinyl phenyl methyl diphenyl dimethicone.

Another class of silicone components suitable for use in a silicone oilphase herein includes polydiorganosiloxane-polyoxyalkylene copolymerscontaining at least one polydiorganosiloxane segment and at least onepolyoxyalkylene segment. Suitable polydiorganosiloxane segments andcopolymers thereof include those described in WO98/22085. Suitablepolydiorganosiloxane-polyalkylene copolymers are available commerciallyunder the trade names Belsil (RTM) from Wacker-Chemie GmbH, Munich, andAbil (RTM) from Th. Goldschmidt Ltd., England, for example Belsil (RTM)6031 and Abil (RTM) B88183. A particularly preferred copolymer fluidblend for use herein includes Dow Corning DC3225C which has the CTFAdesignation Dimethicone/Dimethicone copolyol.

Sunscreens

In still further embodiments, the present invention providescompositions comprising an organic sunscreen. In some embodiments,suitable sunscreens include UVA absorbing properties and/or UVBabsorbing properties. The exact amount of the sunscreen active will varydepending upon the desired Sun Protection Factor (i.e., the “SPF”) ofthe composition, as well as the desired level of UV protection. Thecompositions of the present invention preferably comprise an SPF of atleast 10, preferably at least 15. SPF is a commonly used measure ofphotoprotection of a sunscreen against erythema. The SPF is defined as aratio of the ultraviolet energy required to produce minimal erythema onprotected skin to that required to products the same minimal erythema onunprotected skin in the same individual (See, Fed. Reg., 43, No 166, pp.38206-38269, Aug. 25, 1978). Amounts of the sunscreen used are typicallyfrom about 2% to about 20%, more typically from about 4% to about 14%.Suitable sunscreens include, but are not limited to, those found in theWenninger and McEwen (eds.), CTFA International Cosmetic IngredientDictionary and Handbook, 7^(th) edition, volume 2 pp. 1672 (TheCosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C.,1997).

In some embodiments, compositions of the present invention comprise anUVA absorbing sunscreen actives which absorb UV radiation having awavelength of from about 320 nm to about 400 nm. Suitable UVA absorbingsunscreen actives are selected from dibenzoylmethane derivatives,anthranilate derivatives such as methylanthranilate and homomethyl,1-N-acetylanthranilate, and mixtures thereof. Examples ofdibenzoylmethane sunscreen actives are described in U.S. Pat. No.4,387,089, as well as in Lowe and Shaath (eds), Sunscreens: Development,Evaluation, and Regulatory Aspects, Marcel Dekker, Inc (1990). The UVAabsorbing sunscreen active is preferably present in an amount to providebroad-spectrum UVA protection either independently, or in combinationwith, other UV protective actives which may be present in thecomposition.

Suitable UVA sunscreen actives are dibenzoylmethane sunscreen activesand their derivatives. They include, but are not limited to, thoseselected from 2-methyldibenzoylmethane, 4-methyldibenzoylmethane,4-isopropyldibenzoylmethane, 4-tert-butyldibenzoylmethane,2,4-dimethyldibenzoylmethane, 2,5-dimethyldibenzoyl-methane,4,4′-diisopropylbenzoylmethane,4-(1,1-dimethylethyl)-4′-methoxydiben-zoylmethane,2-methyl-5-isopropyl-4′-methoxydibenzoylmethane,2-methyl-5-tert-butyl-4′-methoxydibenzoylmethane,2,4-dimethyl-4′-methoxydibenzoyl-methane,2,6-dimethyl-4′-tert-butyl-4′methoxydibenzoylmethane, and mixturesthereof. Preferred dibenzoyl sunscreen actives include those selectedfrom 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,4-isopropyldibenzoylmethane, and mixtures thereof. A preferred sunscreenactive is 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane.

The sunscreen active 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,which is also known as butyl methoxydibenzoylmethane or Avobenzone, iscommercially available under the names of PARSOL® 1789 from GivaudanRoure (International) S. A. (Basel, Switzerland) and EUSOLEX® 9020 fromMerck & Co., Inc (Whitehouse Station, N.J.). The sunscreen4-isoproplydibenzoylmethane, which is also known asisopropyldibenzoylmethane, is commercially available from Merck underthe name of EUSOLEX® 8020.

In further embodiments, the compositions of the present inventioncomprise a UVB sunscreen active that absorbs UV radiation having awavelength of from about 290 nm to about 320 nm. The compositionscomprise an amount of the UVB sunscreen active compound which is safeand effective to provide UVB protection either independently, or incombination with, other UV protective actives which may be present inthe compositions. In some embodiments, the compositions comprise fromabout 0.1% to abut 16%, more, preferably from about 0.1% to about 12%,and most preferably from about 0.5% to about 8% by weight, of UVBabsorbing organic sunscreen.

A variety of UVB sunscreen actives are suitable for use herein.Nonlimiting examples of such organic sunscreen actives include thosedescribed in U.S. Pat. No. 5,087,372, U.S. Pat. No. 5,073,371, U.S. Pat.No. 5,073,372, and Segarin et al., Cosmetics Science and Technology, atChapter VIII, pages 189 et seq. Additional useful sunscreens includethose described in U.S. Pat. No. 4,937,370, and U.S. Pat. No. 4,999,186.Preferred UVB sunscreen actives are selected from2-ethylhexyl-2-cyano-3,2-ethylhexyl N,N-dimethyl-p-aminobenzoate,p-aminobenzoic acid, oxybenzone, homomenthyl salicylate, octylsalicylate, 4,4′-methoxy-t-butyldibenzoylmethane, 4-isopropyldibenzoylmethane, 3-benzylidene camphor, 3-(4-methylbenzylidene)camphor, 3-diphenylacrylate (referred to as octocrylene),2-phenyl-benzimidazole-5-sulphonic acid (PBSA), cinnamates and theirderivatives such as 2-ethylhexyl-p-methoxycinnamate andoctyl-p-methoxycinnamate, TEA salicylate, octyldimethyl PABA, camphorderivatives and their derivatives, and mixtures thereof. Preferredorganic sunscreen actives are 2-ethylhexyl-2-cyano-3,3-diphenylacrylate(referred to as octocrylene), 2-phenylbenzimidazole-5-sulphonic acid(PBSA), octyl-p-methoxycinnamate, and mixtures thereof. Salt and acidneutralized forms of the acidic sunscreens are also useful herein.

In some embodiments of the present invention, the compositions furtherinclude an agent useful in stabilizing the UVA sunscreen to prevent itfrom photo-degrading on exposure to UV radiation and thereby maintainingits UVA protection efficacy. A wide range of compounds have been citedas providing these stabilizing properties. It is contemplated that thesecompounds are chosen to complement both the UVA sunscreen and thecomposition as a whole. Suitable stabilizing agents include, but are notlimited to, those described in U.S. Pat. Nos. 5,972,316; 5,968,485;5,935,556; 5,827,508 and WO 00/06110. Preferred examples of stabilizingagents for use in the present invention include2-ethylhexyl-2-cyano-3,3-diphenylacrylate (referred to as octocrylene),ethyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl-3,3-diphenylacrylate,ethyl-3,3-bis(4-methoxyphenyl)acrylate, and mixtures thereof.2-ethylhexyl-2-cyano-3,3-diphenylacrylate is most preferred.

In some embodiments, an agent is added to any of the compositions usefulin the present invention to improve the skin, particularly thosecompositions with enhanced resistance to being washed off by water, orrubbed off. A preferred agent which provides this benefit is a copolymerof ethylene and acrylic acid (See e.g., U.S. Pat. No. 4,663,157).

In addition to the organic sunscreens, in some embodiments, thecompositions of the present invention additionally comprise inorganicphysical sunblocks. Nonlimiting examples of suitable physical sunblocksare described in CTFA International Cosmetic Ingredient Dictionary,6^(th) Edition, 1995, pp. 1026-28 and 1103; and Sayre et al., J. Soc.Cosmet. Chem., 41:103-109 (1990). Preferred inorganic physical sunblocksinclude zinc oxide and titanium dioxide, and mixtures thereof.

When used, the physical sunblocks are present in an amount such that thepresent compositions are transparent on the skin (i.e., non-whitening),preferably less than or equal to about 5%. When titanium dioxide isused, it can have an anatase, rutile, or amorphous structure. Physicalsunblock particles (e.g., titanium dioxide and zinc oxide), can beuncoated or coated with a variety of materials including but not limitedto amino acids, aluminum compounds such as alumina, aluminum stearate,aluminum laurate, and the like; carboxylic acids and their salts eggstearic acid and its salts; phospholipids such as lecithin; organicsilicone compounds; inorganic silicone compounds such as silica andsilicates; and mixtures thereof. A preferred titanium dioxide iscommercially available from Tayca (Japan) and is distributed by Tri-KIndustries (Emerson, N.J.) under the MT micro-ionized series (e.g., MT100SAS). In some embodiments, the compositions of the present inventioncomprise from about 0.1% to about 10%, more preferably from about 0.1%to about 4%, and most preferably from about 0.5% to about 2.5%, byweight, of inorganic sunscreen.

Antimicrobial and Antifungal Actives

In some embodiments, the compositions of the present invention compriseantimicrobial and/or antifungal actives. Non-limiting examples ofantimicrobial and antifungal actives useful herein include, but are notlimited to β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, 2,4,4′-trichloro-2′-hydroxydiphenyl ether, 3,4,4′-trichlorobanilide, phenoxyethanol, phenoxypropanol, phenoxyisopropanol, doxycycline, capreomycin, chlorhexidine,chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidineisethionate, metronidazole, pentamidine, gentamicin, kanamycin,lineomycin, methacycline, methenamine, minocycline, neomycin,netilmicin, paromomycin, streptomycin, tobramycin, miconazole,tetracycline hydrochloride, erythromycin, zinc erythromycin,erythromycin estolate, erythromycin stearate, amikacin sulfate,doxycycline hydrochloride, capreomycin sulfate, chlorhexidine gluconate,chlorhexidine hydrochloride, chlortetracycline hydrochloride,oxytetracycline hydrochloride, clindamycin hydrochloride, ethambutolhydrochloride, metronidazole hydrochloride, pentamidine hydrochloride,gentamicin sulfate, kanamycin sulfate, lineomycin hydrochloride,methacycline hydrochloride, methenamine hippurate, methenaminemandelate, minocycline hydrochloride, neomycin sulfate, netilmicinsulfate, paromomycin sulfate, streptomycin sulfate, tobramycin sulfate,miconazole hydrochloride, amanfadine hydrochloride, amanfadine sulfate,octopirox, parachlorometa xylenol, nystatin, tolnaftate, clotrimazole,cetylpyridinium chloride (CPC), piroctone olamine, selenium sulfide,ketoconazole, triclocarbon, triclosan, zinc pyrithione, itraconazole,asiatic acid, hinokitiol, mipirocin, clinacycin hydrochloride, benzoylperoxide, benzyl peroxide, minocyclin, phenoxy isopropanol, and mixturesthereof, as well as those described in European Patent No. 0 680 745.

Other Optional Ingredients

In some additional embodiments, a variety of optional ingredients suchas neutralizing agents, perfumes, and coloring agents, find use in thecompositions of the present invention. It is preferred that anyadditional ingredients enhance the skin softness/smoothness benefits ofthe product. In addition it is preferred that any such ingredients donot negatively impact the aesthetic properties of the product. Thus,high levels of proteins such as collagen and elastin are typically notpreferred in compositions useful in the present invention.

In some embodiments, the compositions of the present invention alsocontain from about 0.01% to about 10%, preferably from about 0.1% toabout 5% of a panthenol moisturizer. In preferred embodiments, thepanthenol moisturizer is selected from D-panthenol([R]-2,4-dihydroxy-N-[3-hydroxypropyl)]-3,3-dimethylbutamide),DL-panthenol, calcium pantothenate, royal jelly, panthetine,pantotheine, panthenyl ethyl ether, pangamic acid, pyridoxin, andpantoyl lactose.

Neutralizing agents suitable for use in neutralizing acidic groupcontaining hydrophilic gelling agents herein include sodium hydroxide,potassium hydroxide, ammonium hydroxide, monoethanolamine,triethanolamine, amino methyl propanol, tris-buffer and triethanolamine.

Other optional materials include keratolytic agents; water-soluble orsolubilizable preservatives preferably at a level of from about 0.1% toabout 5%, such as Germall 115, methyl, ethyl, propyl and butyl esters ofhydroxybenzoic acid, benzyl alcohol, DMDM hydantoin iodopropanylbutylcarbanate available under the trade name Glydant Plus from Lonza,EDTA, Euxyl (RTM) K400, Bromopol (2-bromo-2-nitropropane-1,3-diol) andphenoxypropanol; anti-bacterials such as Irgasan (RTM) andphenoxyethanol (preferably at levels of from 0.1% to about 5%); solubleor colloidally-soluble moisturising agents such as hylaronic acid andstarch-grafted sodium polyacrylates such as Sanwet (RTM) IM-1000,IM-1500 and IM-2500 available from Celanese Superabsorbent Materials,Portsmith, Va., and described in U.S. Pat. No. 4,076,663; vitamins suchas vitamin A, vitamin C, vitamin E and derivatives thereof and buildingblocks thereof such as phytantriol and vitamin K and components thereofsuch as the fatty alcohol dodecatrienol; alpha and beta hydroxyacids;aloe vera; sphingosines and phytosphingosines, cholesterol; skinwhitening agents; N-acetyl cysteine; coloring agents; antibacterialagents such as TCC/TCS, also known as triclosan and trichlorocarbon;perfumes and perfume solubilizers. Examples of alpha hydroxy acidsinclude glycolic acid, lactic acid, malic acid, citric acid, glycolicacid in conjunction with ammonium glycolate, alpha-hydroxy ethanoicacid, alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid,hydroxycaprylic acid, mixed fruit acid, tri-alpha hydroxy fruit acids,triple fruit acid, sugar cane extract, alpha hydroxy and botanicals,such as those comprising I-alpha hydroxy acid and glycomer incrosslinked fatty acids alpha nutrium. Preferred examples of alphahydroxy acids are glycolic acid and lactic acid. It is preferred thatalpha hydroxy acids are used in levels of up to 10%.

In some embodiments, a safe and effective amount of an anti-inflammatoryagent is added to the compositions of the present invention, preferablyfrom about 0.1% to about 5%, more preferably from about 0.1% to about2%, of the composition. The anti-inflammatory agent enhances the skinappearance benefits of the present invention (e.g., such agentscontribute to a more uniform and acceptable skin tone or colour). Theexact amount of anti-inflammatory agent to be used in the compositionswill depend on the particular anti-inflammatory agent utilized sincesuch agents vary widely in potency.

In further embodiments, compositions of the present invention furtherinclude an anti-oxidant/radical scavenger. The anti-oxidant/radicalscavenger is especially useful for providing protection against UVradiation which can cause increased scaling or texture changes in thestratum corneum and against other environmental agents which can causeskin damage. Suitable amounts are from about 0.1% to about 10%, morepreferably from about 1% to about 5%, of the composition.Anti-oxidants/radical scavengers include compounds such as ascorbic acid(vitamin C) and its salts.

The inclusion of a chelating agent in some embodiments of the presentinvention, is especially useful for providing protection against UVradiation which can contribute to excessive scaling or skin texturechanges and against other environmental agents which can cause skindamage. A suitable amount is from about 0.01% to about 1%, morepreferably from about 0.05% to about 0.5%, of the composition. Exemplarychelators that are useful herein include those described in U.S. Pat.No. 5,487,884. Preferred chelators useful in compositions of the subjectinvention include ethylenediamine tetraacetic acid (EDTA), furildioxime,and derivatives thereof.

In still further embodiments, the compositions of the present inventionalso comprise a skin lightening agent. When used, the compositionspreferably comprise from about 0.1% to about 10%, more preferably fromabout 0.2% to about 5%, also preferably from about 0.5% to about 2%, ofa skin lightening agent. Suitable skin lightening agents include thoseknown in the art, including kojic acid, arbutin, ascorbic acid andderivatives thereof (e.g., magnesium ascorbyl phosphate). Further skinlightening agents suitable for use herein also include those describedin WO 95/34280 and WO 95/23780; each incorporated herein by reference.

Other optional materials include water-soluble or solubilizablepreservatives preferably at a level of from about 0.1% to about 5%, suchas Germall 115, methyl, ethyl, propyl and butyl esters of hydroxybenzoicacid, benzyl alcohol, DMDM hydantoin iodopropanyl butylcarbanateavailable under the trade name Glydant Plus (Lonza), EDTA, Euxyl (RTM)K400, Bromopol (2-bromo-2-nitropropane-1,3-diol) and phenoxypropanol;anti-bacterials such as Irgasan (RTM) and phenoxyethanol (preferably atlevels of from 0.1% to about 5%). Antibacterial agents such as TCC/TCS,also known as triclosan and trichlorocarbon are also useful incompositions of the present invention.

Other optional materials herein include pigments which, whenwater-insoluble, contribute to and are included in the total level ofoil phase ingredients. Pigments suitable for use in the compositions ofthe present invention can be organic and/or inorganic. Also includedwithin the term “pigment” are materials having a low colour or lustersuch as matte finishing agents, and also light scattering agents.Preferably, the compositions of the present invention compriseparticulate materials having a refractive index of from about 1.3 toabout 1.7, the particulate materials being dispersed in the compositionand having a median particle size of from about 2 to about 30 μm.Preferably the particulates useful herein have relatively narrowdistributions, by which is meant that more than 50% of the particlesfall within 3 μm either side of the respective median value. It is alsopreferred that more than 50%, preferably more than 60%, and even morepreferably more than 70% of is particles fall within the size rangesprescribed for the respective median values. Suitable particulatematerials include organic or organosilicone and preferablyorganosilicone polymers. Preferred particles are free-flowing, solid,materials. By “solid” is meant that the particles are not hollow. Thevoid at the center of hollow particles can have an adverse effect onrefractive index and therefore the visual effects of the particles oneither skin or the composition. Suitable organic particulate materialsinclude those made of polymethylsilsesquioxane, referenced above,polyamide, polythene, polyacrylonitrile, polyacrylic acid,polymethacrylic acid, polystyrene, polytetrafluoroethylene (PTFE) andpoly(vinylidene chloride). Copolymers derived from monomers of theaforementioned materials can also be used. Inorganic materials includesilica and boron nitride. Representative commercially available examplesof useful particulate materials herein are Tospearl® 145 which has amedian particle size of about 4.5 μm and EA-209® from Kobo which is anethylene/acrylic acid copolymer having a median particle size of about10 μm, Nylon-12 available under the trade name Orgasol 2002 from ElfAtochem, France, or mixtures thereof.

Further examples of suitable pigments include titanium dioxide,predispersed titanium dioxide from Kobo (e.g., Kobo GWL75CAP), ironoxides, acyglutamate iron oxides, ultramarine blue, D&C dyes, carmine,and mixtures thereof. Depending upon the type of composition, a mixtureof pigments will often find use. The preferred pigments for use hereinfrom the viewpoint of moisturisation, skin feel, skin appearance andemulsion compatibility are treated pigments. The pigments can be treatedwith compounds such as amino acids, silicones, lecithin and ester oils.

Suitably, the pH of the compositions herein is in the range from about6.1 to about 10.0, wherein the pH of the final composition is adjustedby addition of acidic, basic or buffer salts as necessary.

Preparation of Compositions

The compositions of the present invention are prepared by standardtechniques well known to those skilled in the art. In general, theaqueous phase and/or the oil phase are prepared separately, withmaterials of similar phase partitioning being added in any order. If thefinal product is an emulsion, the two phases are then combined withvigorous stirring. Any ingredients in the formulation with highvolatility, or which are susceptible to hydrolysis at high temperatures,can be added with gentle stirring towards the end of the process, postemulsification if applicable.

Proteases with reduced allergenicity/immunogenicity also find use in thetreatment of textiles. “Textile treatment” comprises a process whereintextiles, individual yarns or fibers that can be woven, felted orknitted into textiles or garments are treated to produce a desiredcharacteristic. Examples of such desired characteristics are“stone-washing,” depilling, dehairing, desizing, softening, and othertextile treatments well known to those of skill in the art.

In one embodiment of the present invention, the epitopes identifiedherein are used to elicit an immune response (e.g., where it is desiredto raise antibodies against a protease including one or both of suchepitopes. Such antibodies find use in screening for other proteases thatinclude one or both of these regions, or regions highly homologousthereto. Accordingly, the present invention provides a proteaseincluding one or both of the following sequences: (i) residues 70-84and/or (ii) residues 109-123 of Bacillus amyloliquefaciens subtilisin.The present invention can be embodied in immunoassays utilizing isolatednatural epitope, recombinant protein, or synthetic peptide representingspecific epitopic regions to evaluate persons for sensitization toproteins including these or highly homologous regions.

In another embodiment, the epitopic fragments herein are used in thedetection of antigen presenting cells having MHC molecules capable ofbinding and displaying such fragments. For example, the epitopicfragments can include a detectable label (e.g., radiolabel). The labeledfragments are then incubated with cells of interest, and then cellswhich bind (or display) the labeled fragments are detected.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the identification of CD4⁺T-cell epitopes in subtilisin Carlsberg proteins. The present inventionalso provides for the production of altered peptides which, whenincorporated into a wild-type subtilisin Carlsberg protein produce analtered immunogenic response, preferably a low immunogenic response inhumans. In particular, the present invention provides means, includingmethods and compositions suitable for reducing the immunogenicity ofALCALASE® enzyme. The present invention also provides for the productionof altered peptides which when incorporated into a wild-type subtilisinCarlsberg protein sequence, are no longer capable of initiating the CD4⁺T-cell response or at least reduce the allergic response. In particular,the present invention provides means, including methods and compositionssuitable for reducing the immunogenicity of a wild-type subtilisinCarlsberg.

In one embodiment, the present invention provides T-cell epitopes of theALCALASE® enzyme. These epitopes are provided in various sequences setforth herein (See, FIGS. 2 and 3) and include but are not limited topeptide number 1 (SEQ ID NO.2); peptide number 7 (SEQ ID NO:8); peptidenumber 14 (SEQ ID NO:15); peptide number 29 (SEQ ID NO:30); peptidenumber 39 (SEQ ID NO:40). In another embodiment, the present inventionprovides altered sequences of the identified epitopes which are suitablefor substitution into the ALCALASE® enzyme.

The present invention provides methods for the identification of CD4⁺T-cell epitopes in wild-type subtilisin Carlsberg proteins. Theinvention further provides for the production of peptides that are nolonger capable of initiating the CD4⁺ T-cell response. In particular,the present invention provides means, including methods and compositionssuitable for reducing the immunogenicity of ALCALASE® enzyme.

The development of an antibody to a protein requires a series of eventsthat begin with a peptide segment derived from that protein beingpresented on the surface of an activated antigen presenting cell (APC).The peptide is associated with a specific protein on the surface of theantigen presenting cell, namely a protein in the majorhistocompatibility complex (MHC) (in humans, the MHC is referred to asthe “human leukocyte antigen” (HLA) system). The bound peptide iscapable of interacting with a second cell type, the T-cell.Specifically, the T-cell is of the subtype recognized by the expressionof the CD4 protein on its surface (i.e., CD4⁺ T-cell). If theinteraction is successful, the specific CD4⁺ T-cell grows and divides(i.e., proliferates) and becomes capable of interacting with anantibody-producing cell (i.e., B-cell). If that interaction issuccessful, the B-cell proliferates and becomes a center for theproduction of antibodies that are specifically directed against theoriginal protein. Thus the ultimate production of an antibody isdependent on the initial activation of a CD4⁺ T-cell that is specificfor a single peptide sequence (i.e., an epitope). Using the compositionsand methods described herein, it is possible to predict which peptidesfrom a target protein, such as wild-type subtilisin Carlsberg proteinswill be capable of the initial activation of specific CD4⁺ T-cells.

In some preferred embodiments of the present invention, subtilisinCarlsberg proteins include: i) ALCALASE® enzyme (SEQ ID NO:1); ii)subtilisin Carlsberg proteins having similar catalytic activity toALCALASE® enzyme and having at least about 80%, 85%, 90%, 95%, 97%, 98%or 99% amino acid sequence identity to SEQ ID NO:1, and preferably atleast 90%, at least 95% or at least 97% amino acid sequence identity toSEQ ID NO:1; and iii) variants of subtilisin Carlsberg proteins

Subtilisins are serine proteases (typically, bacterial and fungal) whichgenerally act to cleave peptide bonds. While amino acid sequences of thesubtilisins are not entirely homologous, the subtilisins exhibit thesame or similar type of proteolytic activity and have a common aminoacid sequence defining a catalytic triad which distinguishes them fromthe related class of serine proteases, chymotrypsin. The catalytic triadis, reading from amino to carboxy terminus, aspartate-histidine-serine.The wild-type subtilisin Carlsberg proteins and the modified proteinsherein have this catalytic triad.

Subtilisin Carlsberg proteins having similar catalytic activity to theALCALASE® enzyme as shown in P00780 and FIG. 1 (SEQ ID NO:1), includethose subtilisin Carlsberg proteins obtainable from Bacilluslicheniformis, such as, but not limited to subtilisins having EMBLAccession code X91262, EMBL Accession code X91261, and EMBL Accessioncode X91260. The catalytic domains of these subtilisins have greaterthan 90% amino acid sequence identity with the ALCALASE® enzyme. In apreferred embodiment, these wild-type subtilisin Carlsberg proteins haveat least one and preferably between 1 and 6 significant epitopes incommon with the identified significant ALCALASE® enzyme epitopes of SEQID NO:1.

In some embodiments of the present invention, the variants of subtilisinCarlsberg proteins are naturally occurring (e.g., obtained from Bacilluslicheniformis strains), while in other embodiments, they are geneticallyengineered variants (i.e., recombinant proteins). These variants includerecombinant proteins with alterations in one or more amino acidresidues, wherein the altered amino acid residue is found in a positionother than in a significant epitope. For example, in some embodiments,variants include one or more alterations to an amino acid residuelocated at a position corresponding to positions 1-15, 19-33, 40-54,85-99, and 115-129; of SEQ ID NO:1. In other embodiments, variantsinclude two or more amino acid alterations to amino acids located atpositions corresponding to positions 1-15, 19-33, 40-54, 85-99, and115-129 of SEQ ID NO:1. In still further embodiments, the variantsinclude between 2 and 10 alterations or 2 and 6 alterations to aminoacid residues located at a position corresponding to positions 1-15,19-33, 40-54, 85-99, and 115-129 of SEQ ID NO:1. In some embodiments,the alterations include substitutions, deletions and/or insertions of anamino acid or amino acid sequence, wherein the alteration results in analtered phenotype of the enzyme. For example, in some embodiments, thealteration(s) results in enhanced stability, enhanced enzymaticactivity, enhanced thermal stability, increased alkaline stabilityand/or other desired properties.

Alteration or modification to one or more significant epitopes resultingin a modified wild-type subtilisin Carlsberg protein, which includes amodified ALCALASE or modified variant subtilisin Carlsberg, may includea) modification of the epitope by substitution, deletion or insertion ofone or more amino acids in the epitope or b) modification of the epitopeby substitution with an analogous sequence from a homologous proteinwhich analogous sequence produces a lesser immunogenic response due toT-cell recognition than the parent wild-type subtilisin Carlsberg.

In some embodiments of the present invention, a significant epitope ismodified by the substitution, deletion and/or insertion of at least one,two, three, four, five, six, seven and as many as fifteen amino acidresidues in the epitope. For example, in one preferred embodiment,peptide number 39 (SEQ ID NO:40), corresponding to amino acid residuepositions 115-129 of SEQ ID NO. 1, is altered. The altered amino acidsequence comprises a substitution of one or more positions of 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, and/or 129.In other embodiments, the altered peptide comprises substitution of twoor more positions of 115-129. In other preferred embodiments, peptidenumber 7 (SEQ ID NO: 8; corresponding to amino acid residue positions19-33 of SEQ ID NO:1) is altered. In these embodiments, the alteredamino acid sequence comprises a substitution of one or more positions of19, 20, 21, 22, 23, 24, 25, 26 27 28, 29, 30, 31, 32, and/or 33 (e.g.positions 19-33). Substitutions are made by replacing the wild-typeamino acid residue with a different amino acid. In some preferredembodiments, replacement is preferred using one of the natural L-aminoacids (i.e., Ala, Asn, Asp, Cys, Glu, Gly, Phe, His, Ile, Lys, Leu, Met,Gln, Ser, Thr, Trp, Tyr and Val). In further embodiments, the alteredpeptide is peptide number 7, comprising a deletion of one or more aminoacid residues corresponding to positions 19-33 of SEQ ID NO:1. In stillfurther embodiments, the altered peptide corresponds to peptides 7 and8, with the sequence QGFKGANVKVAVLDTGIQ (SEQ ID NO:90). Thus, variousmodifications in the epitopes of interest are provided which providereduced immunogenicity to the variant subtilisin Carlsberg proteins.

In further embodiments of the invention, a modified wild-type subtilisinCarlsberg protein includes the substitution of an analogous epitopesegment from a homologous protease, wherein the analogous epitopesegment produces a reduced immunogenic response (e.g., a reduced T-cellresponse) compared to the epitope which has been substituted in thewild-type parent. For example in some embodiments, peptide number 7(i.e., corresponding to positions 19-33 of SEQ ID NO:1) is replaced withan analogous segment from another homologous subtilisin such assubtilisin BPN′ from B. amyloliquefaciens or subtilisin 168 from B.subtilis, wherein the analogous segment is not a significant epitope. Insome embodiments, the homologous protease is obtained from a prokaryoticorganism, while in other embodiments, it is obtained from an eukaryoticorganism. Examples of suitable prokaryotic organisms include, but arenot limited to such Gram-negative organisms as E. coli and Pseudomonasspp. and such Gram-positive microorganisms as Micrococcus spp. andBacillus spp.

In some embodiments, the epitopes of the wild-type subtilisin Carlsbergproteins are modified by methods well known in the art. (See e.g.,Zoller et al., Nucl. Acids Res., 10:6487-6500 [1982]; and Yuckenberg etal., (1991), in McPherson (ed.), Directed Mutagenesis: A PracticalApproach, [1991], at pp. 27-48). As indicated above, these modificationsinclude amino acid residue deletion, substitution, and/or insertion. Forexample one or more amino acid residues are modified by site-specificamino acid substitutions. Indeed, commercially available mutagenesiskits find use in producing these variant proteins.

In some embodiments amino acid residues identified for substitution,insertion and/or deletion are conserved residues, whereas in otherembodiments they are not. In preferred embodiments involving residueswhich are not conserved, the replacement of one or more amino acids islimited to substitutions which produce a modified peptide with an aminoacid sequence that does not correspond to one found in nature. In thecase of conserved residues, such replacements do not result in anaturally-occurring sequence.

Cassette mutagenesis also finds use in the present invention tofacilitate the construction of the modified proteins of the presentinvention. According to this method, the naturally-occurring geneencoding the protein is obtained and sequenced in whole or in part.Then, the sequence is scanned for a point at which it is desired to makea mutation (e.g., deletion, insertion or substitution) of one or moreamino acids in the encoded protein. The sequences flanking this pointare evaluated for the presence of restriction sites for replacing ashort segment of the gene with an oligonucleotide pool which whenexpressed will encode various mutants. Such restriction sites arepreferably unique sites within the protein gene so as to facilitate thereplacement of the gene segment. However, any convenient restrictionsite which is not overly redundant in the protein gene finds use withthe present invention, provided that the gene fragments generated byrestriction digestion can be reassembled in proper sequence. Ifrestriction sites are not present at locations within a convenientdistance from the selected point (e.g., from 10 to 15 nucleotides), suchsites are generated by substituting nucleotides in the gene such thatneither the reading frame nor the amino acids encoded are changed in thefinal construction. In some embodiments, mutation of the gene in orderto change its sequence to conform to the desired sequence isaccomplished by M13 primer extension in accord with generally knownmethods. The task of locating suitable flanking regions and evaluatingthe needed changes to arrive at two convenient restriction sitesequences is made routine by the redundancy of the genetic code, arestriction enzyme map of the gene and the large number of differentrestriction enzymes. Note that if a convenient flanking restriction siteis available, the above method need be used only in connection with theflanking region which does not contain a restriction site. However, itis not intended that the present invention be limited to these means, asother suitable methods known to those in the art find use in the presentinvention.

In some embodiments, once the DNA (either naturally-occurring orrecombinant) is cloned, the restriction sites flanking the positions tobe mutated are digested with the cognate restriction enzymes and aplurality of end termini-complementary oligonucleotide cassettes areligated into the gene. The mutagenesis is simplified by this methodbecause all of the oligonucleotides can be synthesized so as to have thesame restriction sites, and no synthetic linkers are necessary to createthe restriction sites.

When a peptide comprising the altered epitope is analyzed in the assayof the present invention, it preferably results in lesser T-cellproliferation than the peptide comprising the wild-type subtilisin. Morepreferably when altered, the epitope produces less than three times thebaseline T-cell proliferation, preferably less than two times thebaseline T-cell proliferation and most preferably less than orsubstantially equal to the baseline T-cell proliferation in a sample.

In some embodiments, the wild-type subtilisin Carlsberg proteins andmodified proteins thereof are screened for proteolytic activityaccording to methods well known in the art. Such methods include, butare not limited to the pNA assay and the dimethyl casein (DMC) assaymethod (Rothgeb et al., J. Am Oil Chem. Soc., 65:806 [1988]).

Application of recombinant DNA technology facilitates the rapidmanipulation of protein or peptide sequences by changing the DNAsequence encoding a protein or peptide (i.e., a protein or peptide ofinterest). Application of this strategy to the gene coding for amodified subtilisin Carlsberg protein, such as modified ALCALASE® enzymefacilitates changing the sequence of the epitopes, such that they are nolonger capable of activating CD4⁺ T-cells. In preferred embodiments,these changes reduce the propensity of a subtilisin Carlsberg to producean antibody-binding antibodies (Bab) and/or neutralizing antibodies(Nab) response in humans. Thus, in particularly preferred embodiments,the present invention provides compositions and methods for theidentification of CD4⁺ T-cell epitopes in the ALCALASE® enzyme proteinsequence and production of peptides which are no longer capable ofinitiating the CD4⁺ T-cell response which, when incorporated throughrecombinant DNA technology, into the ALCALASE® enzyme, are contemplatedto reduce the capability of ALCALASE® enzyme to initiate the productionof antibodies.

Therefore, in certain embodiments, a DNA sequence encoding a modifiedwild-type subtilisin Carlsberg (particularly a modified ALCALASE®enzyme) is introduced into a host cell via an expression vector capableof replicating within the host cell. Those of skill in the art are wellaware of suitable vectors for use in host cells, such as Bacillus hostcells (See e.g. Harwood and Cutting (eds.), Molecular Biological Methodsfor Bacillus, John Wiley & Sons, (1990), at page 92). Transformationtechniques are further described in Chang and Cohen, Mol. Gen. Genet.,168: 11-115 [1979]; and Smith et al., Appl. and Env. Microbiol., 51:634[1986]). In some embodiments, the DNA sequence is directly introducedinto a host cell, without insertion into an expression vector. Suchmethods are well known in the art and include but are not limited tocalcium chloride precipitation, electroporation, naked DNA, etc.

In some embodiments, the modified wild-type subtilisin Carlsbergproteins of the present invention are further isolated and/or purified.This is accomplished by separation techniques known in the art,including but not limited to, ion exchange chromatography, affinitychromatography, hydrophobic separation, precipitation, filtration,microfiltration, gel electrophoresis, and any other suitable method. Inadditional embodiments, once the protein is isolated and/or purified,further constituents are added to the modified proteins to providecompositions of interest.

There are numerous applications for subtilisin Carlsberg proteins,including ALCALASE® enzyme, including but limited to liquid and powereddetergents, textile treatment formulations, conventional cleaningcompositions and personal care compositions. It is readily understoodthat the subtilisins which include the modified epitopes of the presentinvention find use for any purpose in which ALCALASE® enzyme finds use.

Experimental

The following Examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds); ×g (times gravity); Ci (Curies); OD (optical density);Dulbecco's phosphate buffered solution (DPBS); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Klenow (DNA polymeraseI large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethyleneglycol-bis(β-aminoethyl ether) N,N, N′,N′-tetraacetic acid); EDTA(ethylenediaminetetracetic acid); ATCC (American Type CultureCollection, Rockville, Md.); Cedar Lane (Cedar Lane Laboratories,Ontario, Canada); Gibco/Life Technologies (Gibco/Life Technologies,Grand Island, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.);Pharmacia (Pharmacia Biotech, Piscataway, N.J.); Procter & Gamble(Procter and Gamble, Cincinnati, Ohio); Genencor (GenencorInternational, Palo Alto, Calif.); Endogen (Endogen, Wobum, Mass.);Cedarlane (Cedarlane, Toronto, Canada); Dynal (Dynal, Norway); Novo(Novo Industries A/S, Copenhagen, Denmark); Biosynthesis (Biosynthesis,Louisville, Tex.); TriLux Beta, (TriLux Beta, Wallac, Finland);DuPont/NEN (DuPont/NEN Research Products, Boston, Mass.); TomTec(Hamden, Conn.); and Stratagene (Stratagene, La Jolla, Calif.).

EXAMPLE 1 Preparation of Cells Used in the Assay System for theIdentification of Peptide T-Cell Epitopes in ALCALASE® Using HumanT-Cells

Fresh human peripheral blood cells were collected from 92 humans ofunknown exposure status to ALCALASE® enzyme. These cells were tested todetermine antigenic epitopes in ALCALASE®, as described in Example 3.

Peripheral mononuclear blood cells (stored at room temperature, no olderthan 24 hours) were prepared for use as follows: Approximately 30 mls ofa solution of buffy coat preparation from one unit of whole blood wasbrought to 50 ml with Dulbecco's phosphate buffered solution (DPBS) andsplit into two tubes. The samples were underlaid with 12.5 ml of roomtemperature Lymphoprep density separation media (Nycomed; density 1.077g/ml). The tubes were centrifuged for thirty minutes at 600×gravity (g).The interface of the two phases was collected, pooled and washed inDPBS. The cell density of the resultant solution was measured byhemocytometer, as known in the art. Viability was measured by trypanblue exclusion, as known in the art.

From the resulting solution, a differentiated dendritic cell culture wasprepared from the peripheral blood mononuclear cell sample having adensity of 10⁸ cells per 75 ml culture flask in a solution as describedbelow:

-   -   (1) 50 ml of serum free AIM V media (Gibco/BRL) was supplemented        with a 1:100 dilution beta-mercaptoethanol (Gibco/BRL). The        flasks were laid flat for two hours at 37° C. in 5% CO₂ to allow        adherence of monocytes to the flask wall;    -   (2) Differentiation of the monocyte cells to dendritic cells was        performed as follows: nonadherent cells were removed and the        resultant adherent cells (monocytes) combined with 30 ml of AIM        V, 800 units/ml of GM-CSF (Endogen) and 500 units/ml of IL-4        (Endogen); the resulting mixture was cultured for 5 days at        37° C. in 5% CO₂. After the five days of incubation, the        cytokine TNFα (Endogen) was added to 0.2 units/ml, and the        cytokine IL-1α (Endogen) was added to a final concentration of        50 units/ml and the mixture incubated at 37° C. in 5% CO₂ for        two more days.    -   (3) On the seventh day, mitomycin C was added to a concentration        of 50 micrograms/ml in 100 mM EDTA-containing phosphate buffered        saline (PBS) to stop growth of the now differentiated dendritic        cell culture. The solution was incubated for 60 minutes at        37° C. in 5% CO₂. Dendritic cells were dislodged from the        plastic surface by gently tapping the flask. Dendritic cells        were then centrifuged at 600×g for 5 minutes, washed in DPBS and        counted as described above.    -   (4) The prepared dendritic cells were placed into a 96-well        round bottom plate at a concentration of 2×10⁴ cells/well in 100        microliter total volume of AIM V media, per well.

CD4⁺ T cells were prepared from frozen aliquots of the peripheral bloodcell samples used to prepare the dendritic cells, using reagentsprovided by the Dynal CD4⁺ T-cell enrichment kit (Dynal). The resultantCD4⁺ cell solution was centrifuged, resuspended in AIM V media and thecell density was determined using methods known in the art. The CD4⁺T-cell suspension was then resuspended to a count of 2×10⁶ cells/ml inAIM V media to facilitate efficient manipulation of 96-well plates.

EXAMPLE 2 Identification of T-Cell Epitopes in ALCALASE® for Use in theAssay System

Peptides for use in the assay described in Example 3 were prepared basedupon the full-length amino acid sequence (SEQ ID NO:1) of ALCALASE®enzyme, 15mers comprising the entire sequence of ALCALASE® weresynthetically prepared. Consecutive peptides overlapped by 12 aminoacids. A total of 88 peptides (SEQ ID NOS: 2-89) were created, thesequences of which are provided in FIG. 2.

Peptide antigens were prepared as a 2 mg/ml stock solutions in DMSO.First, 0.5 microliters of the stock solution were placed in each well ofthe 96-well plate in which the differentiated dendritic cells werepreviously placed. Then, 100 microliters of the diluted CD4⁺ T-cellsolution as prepared above, were added to each well. Useful controlsincluded diluted DMSO blanks, and tetanus toxoid positive controls.

The final concentrations in each well, at 20 microliter total volumewere as follows:

-   -   2×10⁴ CD4⁺ T-cells    -   2×10⁵ dendritic cells (R:S of 10:1)    -   5 μM peptide

EXAMPLE 3

Assay for the Identification of Peptide T-Cell Epitopes in ALCALASE®Enzyme Using Human T-Cells

Once the assay reagents (i.e., cells, peptides, etc.) were prepared anddistributed into the 96-well plates, the assays were conducted. Controlsincluded dendritic cells plus CD4+ T-cells alone (with DMSO carrier) andwith tetanus toxoid (Wyeth-Ayerst, Philadelphia, Pa.), at approximately5 Lf/mL.

Cultures were incubated at 37° C. in 5% CO₂ for 5 days. Tritiatedthymidine (NEN) was added at 0.5 microCi/well. The cultures wereharvested and assessed for incorporation the next day, using the WallacTriBeta scintillation detection system.

All of the tests were performed at least in duplicate. All of the testsreported displayed robust positive control responses to the antigentetanus toxoid. Responses were averaged within each experiment, thennormalized to the baseline response. A positive event (i.e., aproliferative response) was recorded if the response was at least 2.95times the baseline response.

The immunogenic responses (i.e., T-cell proliferation) to the preparedpeptides from ALCALASE® enzyme were tallied for all 92 donors and areillustrated in FIG. 3. Six significant epitopes were identified.Peptides identified as being of particular interest include peptides 1,7 and 8, 14, 29 and 39, as indicated in FIGS. 2 and 3. These is peptidescorrespond to the following sequences: SEQ ID Peptide # Sequence NO:  1AQTVPYGIPLIKADK 2 7-8 QGFKGANVKVAVLDTGIQ 90 14 PDLNVVGGASFVAGE 15 29PSVSLYAVKVLNSSG 30 39 TNGMDVINMSLGGPS 40

The overall background rate of responses to this peptide set was2.35±2.56%, for the donors tested. In the above Table, peptide 7-8 is acontinuous sequence of 18 amino acids (peptide #7, please the 3 uniqueamino acids contributed by peptide #8). This combination of peptides 7and 8 was combined due to the good responses observed for both peptides.

EXAMPLE 4 Assay of Variant Peptides

Peptide numbers 7 and 29 are selected for further analysis. Sets ofaltered peptides are constructed based on the sequences of peptidenumber 7 and peptide number 29, wherein the amino acid residues aremodified from the parent sequence. This can be accomplished by acommercial vendor such as Mimostopes (San Diego). An alanine scan isperformed for each peptide (See e.g., Harris, et al., Immunol.,84:555-561 -[1995]; and Maillere et al., Mol. Immunol., 32:1073-1080[1995]). The assay is performed as described in Example 3, utilizing thealtered peptides on a set of donor samples. Proliferative responses arecollated.

In one embodiment, an altered peptide is considered useful for creatinga hypoallergenic protein molecule (i.e., a modified subtilisin proteinaccording to the invention) if at least one and preferably all three ofthe following criteria are met: (1) all donors who respond with astimulation index (SI) of about greater than 2.95 to the parent peptiderespond to the altered peptide with an SI of about 1.0 or less; (2) alldonors who respond weakly to the parent peptide (with an SI greater thanabout 1.0 but less than about 2.95) respond to the altered peptide withan SI of about 1.0 or less; and (3) all non-responders to the parentpeptide are also non-responders to the altered peptide. SI is a measureof the T-cell profilerative response of a peptide compared to a controlpeptide. A SI is calculated for each donor for each peptide.

EXAMPLE 5 HLA Association with an Epitope Peptide Number

The HLA-DR and DQ expression of all the donors tested in both rounds ofassay testing described above are assessed using a commerciallyavailable PCR-based HLA typing kit (Bio-Synthesis). In some embodiments,the phenotypic frequencies of individual HLA-DR and -DQ antigens amongresponders and non-responders to a peptide number are tested using achi-squared analysis with 1 degree of freedom. The increased ordecreased likelihood of reacting to an epitope corresponding to thepeptide number is calculated wherever the HLA antigen in question ispresent in both responding and non-responding donor samples and thecorresponding epitope is considered an HLA associated epitope.

The magnitude of the proliferative response to an individual peptide inresponders and non-responders expressing epitope-associated HLA allelesmay also be analyzed. An “individual responder to the peptide” isdefined by a stimulation index of greater than 2.95. It is contemplatedthat the proliferative response in donors who express an epitopeassociated with HLA alleles would be higher than in peptide responderswho do not express the associated allele.

From the above, it is clear that the present invention provides methodsand compositions for the identification of T-cell epitopes in wild-typesubtilisin Carlsberg, such as ALCALASE®. Once antigenic epitopes areidentified, the epitopes are modified as desired, and the peptidesequences of the modified epitopes incorporated into a wild-typesubtilisin Carlsberg, so that the modified sequence is no longer capableof initiating the CD4⁺ T-cell response or wherein the CD4⁺ T-cellresponse is significantly reduced in comparison to the wild-type parent.In particular, the present invention provides means, including methodsand compositions suitable for reducing the immunogenicity of ALCALASE®.

EXAMPLE 6 Hydrolysis of Dimethyl Casein (“DMC”) by Mutant VariantSubtilisin

Mutant variant subtilisins, isolated and purified by the methodsdescribed herein, are analyzed for their ability to hydrolyze acommercial synthetic substrate, di-methyl casein (Sigma C-9801). A 5mg/ml DMC substrate solution is prepared in the appropriate buffer (5mg/ml DMC, 0.005% (w/w) Tween 80® (polyoxyethylene sorbitan mono-oleate,Sigma P-1754)). Appropriate DMC substrate buffers are prepared (e.g., 50mM sodium acetate for pH 5.5; 50 mMN-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (“TES”) for pH6.5; 50 mM piperazine-N-′-bis-2-ethane sulfonic acid (“PIPES”) for pH7.5; and 50 mM Tris for pH 8.5). To begin testing, 200 μl of the desiredpH substrate are placed into the wells of a microtiter plate (e.g., a 96well plate) and pre-incubated at 37° C. for twenty minutes prior toenzyme addition. A 2,4,6-trinitrobenzene sulfonate salt (“TNBS”) colorreaction method is used to determine activity on a Spectra Max 250spectrophotometer. This assay measures the enzymatic hydrolysis of DMCinto peptides containing free amino groups. These amino groups reactwith 2,4,6-trinitro-benzene sulfonic acid to form a yellow coloredcomplex.

Thus, the more deeply colored the reaction, the more activity ismeasured. The TNBS detection assay can be performed on the supernatantafter two hours of incubation at 37° C. A 1 mg/ml solution of TNBS isprepared in a solution containing 2.4 g NaOH, 45.4 g Na₂B₄)₇.10H₂Odissolved by heating in 1000 ml. From this solution, 60 μl are aliquotedinto a 96-well microtiter plate. Then, 10 μl of the incubated enzymesolution described above is added to each well and mixed for 20 minutesat room temperature. Then, 20 μl of NaH₂PO₄ solution (70.4 g NaH₂PO₄.H₂Oand 1.2 g Na₂SO₃ in 2000 ml) are mixed for 1 minute in the wells to stopthe reaction and the absorbance at 405 nm in a SpectraMax 250spectrophotometer is determined. A blank (same TNBS solution, butwithout the enzyme) is also be prepared and tested. The hydrolysis ismeasured by the following formula:Absorbance₄₀₅ (Enzyme solution)—Absorbance₄₀₅ (without enzyme)at varying enzyme concentrations (0, 2.5, 5, 7.5, and 10 ppm). Thecomparative ability of the mutant variants to hydrolyze such substrateversus subtilisins from a known mutant variant (e.g. a characterizedmutant subtilisin) can be determined in this manner.

EXAMPLE 7 Hydrolysis of Collagen, Elastin, and Keratin by VariantSubtilisin Carlsberg Enzymes

Mutant variant subtilisin Carlsberg enzymes isolated and purified by themethods described above, are often analyzed for their ability tohydrolyze commercial substrates, for example bovine collagen (SigmaC-9879), bovine elastin (Sigma E-1625), and/or bovine keratin (ICNBiomedical 902111). A 5 mg/ml substrate solution is prepared (in 0.005%Tween 80®). Each substrate is prepared in the appropriate pH as known inthe art (e.g., pH 5.5, 6.5, 7.5, and 8.5). To test, 1.5 ml of the eachsubstrate is transferred into 24-well Costar plate at 37° C. The platesare pre-incubated at 37° C. for twenty minutes prior to enzyme addition.A TNBS detection assay as described above is performed on thesupernatant after two hours of incubation at 37° C.

It is contemplated that these assays will find use in demonstrating thecomparative ability of the mutant variants to hydrolyze such substratesversus subtilisins from a known mutant variant. In most case, it iscontemplated that the mutated enzymes will typically show significanthydrolysis of collagen, elastin and keratin substrates at different pHsand different enzyme concentrations, as compared to each other andwild-type enzyme.

EXAMPLE 8 Thermal Stability of Protein Variants inpiperazine-N-′-bis-2-ethane sulfonic acid (“PIPES”) Buffer

In these experiments, the thermal stability of the protein (e.g.,subtilisin Carlsberg) variants in PIPES is determined. Typically, thesedeterminations are conducted using a PCR thermocycler of the typeStratagene Robocycler. The stability of 5.0 ppm enzyme 5.0 ppm enzyme(e.g., the mutants of interest and control mutant enzyme(s)) are testedat five timepoints (e.g., 5, 10, 20, 40, and 60 minutes) at pH 6.5, foreach temperature. For example, the samples are tested at two degreeintervals ranging from 42-56° C., and at every other degree attemperatures ranging from 42-56° C., in the PCR thermocycler gradient.In these experiments, a 50 mM PIPES buffer is prepared (50 mM PIPES,0.005% Tween 80®). Typically, the pH is adjusted to 6.5. However, it isnot intended that the present invention be limited to this particularmethod, as various methods are known in the art to determine the thermalstability of enzymes.

Samples are assayed using standard succinyl-ala-ala-pro-phe-para-nitroanilide (“SAAPFpNA”) assay (See e.g., Delmar, Anal. Biochem., 94:316-320[1979]; and Achtstetter, Arch. Biochem. Biophys., 207:445-54 [1981]), atpH 6.5, and at 25° C. The samples are diluted to about 300milliOD/minute. The thermal stability is typically expressed as enzymehalf-life (min) as determined by:H.L.=ln 2/slope, wherein the slope is the slope of curve of rate v. timefor each temperature.

By using these means, the stability of mutant variants can be readilycompared relative the control mutant enzymes and/or wild-type enzyme.

EXAMPLE 9 Thermal Stability of Subtilisin Carlsberg Variants inN-tris(Hydroxymethyl)methyl-2-Aminoethanesulfonic Acid (“TES”)

In these experiments, the thermal stability of the variants in TES isdetermined. As is described above, 5.0 ppm enzyme (e.g., the mutants ofinterest and the controls) are tested at five timepoints (e.g., 5, 10,20, 40, and 60 minutes) at pH 6.5, for each temperature. For example,the samples are tested at two degree intervals ranging from 42-56° C.,and at every other degree at temperatures ranging from 42-56° C., in thePCR thermocycler gradient. A TES buffer is prepared by mixing 50 mM TES(Sigma T 1375), 0.005% Tween 80®. Typically, the pH is adjusted to 6.5.

Thermal stability of the variants can be determined as activity of theresidual variant as measured using asuccinyl-ala-ala-pro-phe-para-nitroanilide (“AAPFpNA”) as known in theart, using reagents such as Sigma no. S-7388 (mol. wt. 624.6 g/mole)(See e.g., Delmar et al., Anal. Biochem., 94:316-320 [1979]; andAchtstetter, Arch. Biochem. Biophys., 207:445-454 [1981]), tested at pH6.5, and at a temperature of 25° C. The (yellow) p-nitronanilide (pNA)formed in the reaction is measured spectrophotometrically at 410 nm:.ε_(M)=8,480 M⁻¹. cm⁻¹, ( ) with a SpectraMax 250 spectrophotometer, thesamples being diluted to about 300 mOD/min. The thermal stability isexpressed as enzyme half-life (min) as described above. As indicatedabove, these experiments provide means to compare the stability of thevariant enzyme preparations with the control mutant enzyme(s) and/orwild-type enzyme.

EXAMPLE 10 Stability of Subtilisin Carlsberg Variants in BodywashSolutions and Other Personal Care Products

The stability of various subtilisin Carlsberg variants is measured usingthe following protocol.

Method to Measure Solution Stability

In these experiments, subtilisin Carlsberg and mutant variants aretested in at least two studies, with the first study involving testingfor 30 minutes at 45° C., and the second involving testing for 30minutes at 50° C. For these tests, 50/50 (w/w) bodywash solutions areprepared by mixing a commercially available bodywash (e.g., the bodywashsold under the trademark ZEST®, from Procter & Gamble), with deionizedwater. The pH of the buffer blend is approximately 6.8.

The enzymes to be tested are diluted such that their final enzymeconcentration in a 50 w/w % BodyWash: deionized water solution producesa change in OD₄₀₅ Of 0.5 to 1.0 is when 10 μl of the enzyme/body washsolution is assayed using SAAPFpNA assay endpoint method. Once theamount of dilution is ascertained, 200 μl of the diluted mixture isplaced into 96 well microtiter plate wells. The plate are sealed andplaced in a water bath at 40° C., for one study, and at 50° C., for thesecond study. The plates are removed from the water bath after thedesired length of time (e.g., 30 or 45 minutes) and 10 μl samplesassayed by the endpoint method. The percent of activity remaining iscalculated as 100 times the final activity divided by the initialactivity.

In some experiments, the variants including the specific residuesdetermined by the assay of the earlier described example show anincreased amount of enzymatic activity remaining and thus have a broaderthermal stability than the controls. For example, at 50° C., somevariant compounds have a greater percentage activity remaining whereasthe control mutant enzyme and/or the wild-type enzyme without thestabilizing residue variants have a lower percentage of activityremaining. In some experiments, all enzymes have enhanced stability inthe presence of bodywash at 500, but control mutant enzyme-[epitopicvariants] with different stability variants have even better stability.

Indeed, there are numerous applications in which the variant subtilisinCarlsberg enzymes of the present invention that have reducedimmunogenicity find use. In addition to detergents and other cleaningpreparations, the variant subtilisin Carlsberg enzymes having reducedimmunogenicity also find use in personal care products. The followingtables provide the compositions of various products suitable for use intesting. In these tables, the term “minors” encompasses pH modifiers,preservatives, viscosity modifiers, and perfumes. In these tables, theamounts represent approximate weight percent (as provided by themanufacturer), unless otherwise indicated, and are not intended toindicate significant digits. MOISTURISING BODYWASH pH = 7 RAW MATERIALAmount Deionized Water QS Glycerin 4.0 PEG-6 Caprylic/Capric Glycerides4.0 Palm Kernal Fatty acids 3.0 Sodium Laureth-3 Sulphate 45.0 CocamideMEA 3.0 Sodium Lauroamphoacetate 25.0 Soybean Oil 10.0 Polyquaternium-10(JR30M) 0.70 Protease 1000 ppm

BODYWASH pH 6.5 pH 7 pH 8.5 RAW MATERIAL Amount Amount Amount Deionizedwater QS QS QS Sodium Laureth Sulphate 12 15 8 Cocamidopropyl Betaine 810 15 APG Glucoside (Plantacare 2000¹) 0 2 1 Polyquaternium-10 (JR30M)0.25 0 0 Polyquaternium-7 (Mackam 55). 0 0 0.7 Protease 250 ppm 500 ppm1000 ppm¹Cognis

BODY LOTION pH 7 pH 7 pH 7.5 pH 7 RAW MATERIAL Amount Amount AmountAmount DEIONISED WATER QS QS QS QS GLYCERINE 8 8 10 12 ISOHEXADECANE 3 33 6 NIACINAMIDE 0 3 5 6 ISOPROPYL 3 3 3 3 ISOSTEARATE Polyacrylamide, 33 3 3 Isoparaffin, Laureth-7 (Sepigel 305²) PETROLATUM 4 4 4 2 NYLON 122 2 2.5 2.5 DIMETHICONE (DC1403⁴) 2 2 2.5 2.5 SUCROSE 1.5 1.5 1.5 1.5POLYCOTTONSEED OIL Stearyl Alcohol 97% 1 1 1 1 D PANTHENOL 1 1 1 1DL-alphaTOCOPHEROL 1 1 1 1 ACETATE Cetyl Alcohol 95% 0.5 0.5 0.5 1BEHYNYL ALCOHOL 1 1 1 0.5 EMULGADE PL 68/50 0.4 0.4 0.5 0.5 STEARIC ACID0.15 0.15 0.15 0.15 Peg-100-stearate (MYRJ 59¹) 0.15 0.15 0.15 0.15Protease 50 ppm 50 ppm 250 ppm 1000 ppm¹Uniqema²Seppic⁴Dow Corning

ULTRA-HIGH MOISTURISING FACIAL CREAM/LOTION pH 7 pH 7 RAW MATERIALAmount Amount Deionized water QS QS Glycerin 12 5 PEG 400⁶ 0 10Niacinamide 5 7 Isohexadecane 5 5 Dimethicone (DC1403³) 3 2Polyacrylamide, Isoparaffin, Laureth- 3 3 7 (Sepigel 305¹) IsopropylIsostearate 2 2 Polymethylsilsesquioxane 2 2 Cetyl Alcohol 95% 1 1Sucrose polycottonseed oil 1 1 D-Panthenol 1 1 Vitamin E (TocopherolAcetate) 1 1 Stearyl Alcohol 95% 0.5 0.5 Cetearyl Glucoside 0.5 0.5Titanium dioxide 0.3 0.3 Stearic Acid 0.15 0.15 PEG-100-Stearate (Myrj59⁴) 0.15 0.15 Protease 500 ppm 500 ppm¹Seppic³Dow Corning⁴Uniqema⁵Scher Chemicals⁶Dow Chemicals

FACIAL MOISTURIZING CREAM pH 7 pH 7 pH 7.5 RAW MATERIAL Amount AmountAmount Deionized water QS QS QS Glycerin 3 5 10 Petrolatum 3 3 0 CetylAlcohol 95% 1.5 1.5 1 Dimethicone Copolyol 2 2 2 (DC 3225C⁴) IsopropylPalmitate 1 1 0.5 Carbomer 954² 0.7 0.7 0.7 Dimethicone (DC 200/350cs⁴)1 1 1 Stearyl Alcohol 97% 0.5 0.5 1 Stearic acid 0.1 0.1 0.1Peg-100-Stearate (MYRJ 59¹) 0.1 0.1 0.1 Titanium Dioxide 0.3 0.3 0.3Protease 50 ppm 250 ppm 1000 ppm¹Uniqema²BF Goodrich⁴Dow Corning

EXAMPLE 11 Cleaning Compositions

In addition to the compositions described above, the present inventionprovides means to develop cleaning compositions having particularcharacteristics. Indeed, the present invention provides various cleaningcompositions that comprise modified proteases (e.g., subtilisinCarlsberg). In particularly preferred embodiments, an effective amountof one or more protease enzymes described above are included incompositions useful for cleaning a variety of surfaces in need ofproteinaceous stain removal. Such cleaning compositions includedetergent compositions for cleaning hard surfaces; detergentcompositions for cleaning fabrics; dishwashing compositions; oralcleaning compositions; and denture cleaning compositions. It is intendedthat these compositions be provided in any form suitable for theparticular intended use. Preferably, the cleaning compositions of thepresent invention comprise from about 0.0001% to about 10% of one ormore protease enzymes, more preferably from about 0.001% to about 1%,and more preferably still from about 0.001% to about 0.1%. Severalexamples of various cleaning compositions wherein the protease enzymesfind use are discussed in further detail below. All parts, percentagesand ratios used herein are by weight unless otherwise specified.

A. Cleaning Compositions for Hard Surfaces, Dishes, and Fabrics

The protease enzymes (e.g., the variant subtilisin Carlsberg enzymes) ofthe present invention find use in any detergent composition where highsudsing and/or good insoluble substrate removal are desired. Thus, theprotease enzymes of the present invention find use with variousconventional ingredients to provide fully-formulated hard-surfacecleaners, dishwashing compositions, fabric laundering compositions andthe like. These compositions are suitable for use in any form (e.g.,liquid, granules, bars, etc.) acceptable for the particular application.In addition, these compositions are also suitable for use incommercially available “concentrated” detergents which contain as muchas 30%-60% by weight of surfactants.

In some embodiments, the cleaning compositions contain various anionic,nonionic, zwitterionic, etc., surfactants. Such surfactants aretypically present at levels of from about 0.1% to about 60%, preferablyfrom about 1% to about 35%, of the compositions. Suitable surfactantsinclude, but are not limited to the conventional C₁₁-C₁₈ alkyl benzenesulfonates and primary and random alkyl sulfates, the C₁₀-C₁₈ secondary(2,3)alkyl sulfates of the formulas CH₃ (CH₂)x(CHOSO₃).sup.−M.sup.+)CH₃,and CH₃ (CH₂)y(CHOSO₃.sup.−M.sup.+)CH₂CH₃, wherein x and (y+1) areintegers of at least about 7, preferably at least about 9, and M is awater-solubilizing cation, especially sodium, the C₁₀-C₁₈ alkyl alkoxysulfates (especially EO 1-7 ethoxy sulfates), C₁₀-C₁₈ alkyl alkoxycarboxylates (especially the EO 1-7 ethoxycarboxylates), the C₁₀-C₁₈alkyl polyglycosides, and their corresponding sulfated polyglycosides,C₁₂-C₁₈ alpha-sulfonated fatty acid esters, C₁₂-C₁₈ alkyl and alkylphenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy),C₁₂-C₁₈ betaines and sulfobetaines (“sultaines”), C₁₀-C₁₈ amine oxides,C₈-C₂₄ sarcosinates (especially oleoyl sarcosinate), and the like. Thealkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) arepreferred herein. Furthermore, use of such surfactants in combinationwith the aforesaid amine oxide and/or betaine or sultaine surfactants isalso preferred, depending on the desires of the formulator. Otherconventional useful surfactants are known to those in the art,including, but not limited to the particularly useful surfactants suchas the C₁₀-C₁₈ N-methyl glucamides (See, U.S. Pat. No. 5,194,639).

In some embodiments, the compositions of the present invention comprisemember(s) of the class of nonionic surfactants which are condensates ofethylene oxide with a hydrophobic moiety to provide a surfactant havingan average hydrophilic-lipophilic balance (HLB) in the range from 5 to17, preferably from 6 to 14, more preferably from 7 to 12. Thehydrophobic (lipophilic) moiety may be aliphatic or aromatic in natureand the length of the polyoxyethylene group which is condensed with anyparticular hydrophobic group can be readily adjusted to yield awater-soluble compound having the desired degree of balance betweenhydrophilic and hydrophobic elements. Especially preferred are theC₉-C₁₅ primary alcohol ethoxylates (or mixed ethoxy/propoxy) containing3-8 moles of ethylene oxide per mole of alcohol, particularly theC₁₄-C₁₅ primary alcohols containing 6-8 moles of ethylene oxide per moleof alcohol, the C₁₂-C₁₅ primary alcohols containing 35 moles of ethyleneoxide per mole of alcohol, and mixtures thereof.

A wide variety of other ingredients useful in detergent cleaningcompositions find use in the compositions herein, including other activeingredients, carriers, hydrotropes, processing aids, dyes or pigments,solvents for liquid formulations, etc. For an additional increment ofsudsing, suds boosters such as the C₁₀-C₁₆ alkolamides can beincorporated into the compositions, typically at about 1% to about 10%levels. The C₁₀-C₁₄ monoethanol and diethanol amides illustrate atypical class of such suds boosters. Use of such suds boosters with highsudsing adjunct surfactants such as the amine oxides, betaines andsultaines noted above is also advantageous. If desired, solublemagnesium salts such as MgCl₂, MgSO₄, and the like, can be added atlevels of, typically, from about 0.1% to about 2%, to provide additionalsudsing.

The liquid detergent compositions herein typically contain water andother solvents as carriers. Low molecular weight primary or secondaryalcohols (e.g., methanol, ethanol, propanol, and isopropanol) aresuitable. Monohydric alcohols are preferred for solubilizingsurfactants, but polyols such as those containing from about 2 to about6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g.,1,3-propanediol, ethylene glycol, glycerine, and 1,2-propanediol) alsofind use in the detergents of the present invention. In someembodiments, the compositions contain about 90%, or from about 10% toabout 50% of such carriers.

The detergent compositions herein are preferably formulated such thatduring use in aqueous cleaning operations, the wash water has a pHbetween about 6.8 and about 11.0. Thus, finished products are typicallyformulated at this range. Techniques for controlling pH at recommendedusage levels include the use of buffers, alkalis, acids, etc., and arewell known to those skilled in the art.

When formulating the hard surface cleaning compositions and fabriccleaning compositions of the present invention, the formulator may wishto employ various builders at levels from about 5% to about 50% byweight. Typical builders include the 1-10 micron zeolites,polycarboxylates such as citrate and oxydisuccinates, layered silicates,phosphates, and the like. Other conventional builders are known to thosein the art and are suitable for inclusion in the compositions of thepresent invention.

Likewise, the formulator may wish to employ various additional enzymes,such as cellulases, lipases, amylases, peroxidases, and proteases insuch compositions, typically at levels of from about 0.001% to about 1%by weight. Various detersive and fabric care enzymes are well-known inthe laundry detergent art and are suitable for inclusion in thecompositions of the present invention.

Various bleaching compounds, such as the percarbonates, perborates andthe like, also find use in the compositions of the present invention.These bleaching compounds are typically present at levels from about 1%to about 15% by weight. If desired, such compositions can also containbleach activators such as tetraacetyl ethylenediamine,nonanoyloxybenzene sulfonate, and the like, which are also known in theart. Usage levels of such compounds typically range from about 1% toabout 10% by weight.

Various soil release agents, especially of the anionic oligoester type,various chelating agents, especially the aminophosphonates andethylenediaminedisuccinates, various clay soil removal agents,especially ethoxylated tetraethylene pentamine, various dispersingagents, especially polyacrylates and polyasparatates, variousbrighteners, especially anionic brighteners, various dye transferinhibiting agents, such as polyvinyl pyrrolidone, various sudssuppressors, especially silicones and secondary alcohols, various fabricsofteners, especially smectite clays and clay floculating polymers(e.g., poly(oxy ethylene)), and the like all find use in thecompositions of the present invention, most typically at levels rangingfrom about 1% to about 35% by weight.

Enzyme stabilizers also find use in the cleaning compositions of thepresent invention. Such enzyme stabilizers include, but are not limitedto propylene glycol (preferably from about 1% to about 10%), sodiumformate (preferably from about 0.1% to about 1%) and calcium formate(preferably from about 0.1% to about 1%).

1. Hard Surface Cleaning Compositions

In preferred embodiments, hard surface cleaning compositions of thepresent invention comprise an effective amount of one or more proteaseenzymes (e.g., variant subtilisin Carlsberg enzymes), preferably fromabout 0.0001% to about 10%, more preferably from about 0.001% to about5%, more preferably still from about 0.001% to about 1% by weight ofactive protease enzyme of the composition. In addition to comprising oneor more protease enzymes, such hard surface cleaning compositionstypically comprise a surfactant and a water-soluble sequesteringbuilder. However, in certain specialized products such as spray windowcleaners, the surfactants are sometimes not used since they may producea filmy/streaky residue on the glass surface.

The surfactant component, when present, may comprise as little as 0.1%of the compositions herein, but typically the compositions will containfrom about 0.25% to about 10%, more preferably from about 1% to about 5%of surfactant.

Typically the compositions will contain from about 0.5% to about 50% ofa detergency builder, preferably from about 1% to about 10%. Preferably,the pH should be in the range of about 8 to 12. Conventional pHadjustment agents such as sodium hydroxide, sodium carbonate orhydrochloric acid can be used if adjustment is necessary.

In some embodiments, at least one solvent is included in thecompositions. Useful solvents include, but are not limited to, glycolethers such as diethyleneglycol monohexyl ether, diethyleneglycolmonobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycolmonohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycolmonobutyl ether, and diols such as 2,2,4-trimethyl-1,3pentanediol and2-ethyl-1,3-hexanediol. When used, such solvents are typically presentat levels of from about 0.5% to about 15%, preferably from about 3% toabout 11%.

Additionally, highly volatile solvents such as isopropanol or ethanolfind use in the present compositions, in order to facilitate fasterevaporation of the composition from surfaces when the surface is notrinsed after “full strength” application of the composition to thesurface. When used, volatile solvents are typically present at levels offrom about 2% to about 12% in the compositions.

The hard surface cleaning composition embodiment of the presentinvention is illustrated by the following nonlimiting examples. In thefollowing examples, reference to “Protease #” in the examples is to thevariant useful in the present invention compositions having a reducedimmunogenic responding protease variant of percentages of 0.10, 0.20,0.10, 0.05, 0.03, and 0.02. Liquid Hard Surface Cleaning CompositionsExample No. Component 1 2 3 4 5 6 EDTA** 2.90 2.90 Na Citrate 2.90 2.90NaC₁₂ Alkyl- 1.95 1.95 1.95 benzene NaC₁₂ 2.20 2.20 2.20 AlkylsulfateNaC₁₂ 2.20 2.20 2.20 (ethoxy)*** C₁₂ 0.50 0.50 0.50 Dimethylamine NaCumene 1.30 1.30 1.30 sulfonate Hexyl 6.30 6.30 6.30 6.30 6.30 6.30Carbitol*** Water**** Balance to 100%**Na₄ Ethylenediamine diacetic acid***Diethyleneglycol monohexyl ether****All formulae adjusted to pH 7.

In some embodiments of the above examples, additional proteases usefulin the present invention (e.g., variant subtilisin Carlsberg enzymes)are substituted with substantially similar results. In addition, in someembodiments of the above examples, any combination of the reducedimmunogenic proteases useful in the present invention (e.g., variantsubtilisin Carlsberg enzymes) is substituted in the above formulationswith substantially similar results.

The following Table provides sample compositions suitable for cleaninghard surfaces and removing mildew. The product compositions aretypically at approximately pH 7. Spray Compositions for Cleaning HardSurfaces and Removing Household Mildew Example No. Component 7 8 9 10 1112 Protease # 0.20 0.05 0.10 0.30 0.20 0.30 Protease #+14 0.30 0.10Sodium octyl 2.00 2.00 2.00 2.00 2.00 2.00 sulfate Sodium 4.00 4.00 4.004.00 4.00 4.00 dodecyl sulfate NaOH 0.80 0.80 0.80 0.80 0.80 0.80Silicate (Na) 0.04 0.04 0.04 0.04 0.04 0.04 Perfume 0.35 0.35 0.35 0.350.35 0.35 Water Balance to 100%

In these Examples, any combination of the protease enzymes useful in thepresent invention (e.g., variant subtilisin Carlsberg enzymes) issubstituted in with substantially similar results.

2. Dishwashing Compositions

In additional embodiments of the present invention, dishwashingcompositions comprising one or more protease enzymes (e.g., mutantsubtilisin Carlsberg enzymes) are provided. Preferred dishwashingcomposition embodiments of the present invention are illustrated below.Proteases are included with percentages at 0.5, 0.4, 0.1, 0.05, 0.03,and 0.02. In these compositions, the product pH is adjusted to 7.Dishwashing Compositions Example No. Component 13 14 15 16 17 18 C₁₂-C₁₄N- 0.90 0.90 0.90 0.90 0.90 0.90 methyl- C₁₂ ethoxy (1) 12.0 12.0 12.012.0 12.0 12.0 sulfate 2-methyl 4.50 4.50 4.50 4.50 undecanoic acid C₁₂ethoxy (2) 4.50 4.50 4.50 4.50 4.50 4.50 carboxylate C₁₂ alcohol 3.003.00 3.00 3.00 3.00 3.00 ethoxylate (4) C₁₂ amine oxide 3.00 3.00 3.003.00 3.00 3.00 Sodium cumene 2.00 2.00 2.00 2.00 2.00 2.00 sulfonateEthanol 4.00 4.00 4.00 4.00 4.00 4.00 Mg Supp⁺⁺ 0.20 0.20 0.20 0.20 0.200.20 (MgCl₂) Ca Supp⁺⁺ 0.40 0.40 0.40 0.40 0.40 0.40 (CaCl₂) WaterBalance to 100%

In some embodiments of the immediately above examples the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) are substituted in the above formulations, with substantiallysimilar results. Furthermore, in some embodiments of the immediatelyabove examples, any combination of the protease enzymes useful in thepresent invention (e.g., variant subtilisin Carlsberg enzymes), amongothers, is substituted in the above formulations with substantiallysimilar results. Granular Automatic Dishwashing Compositions ExampleComponent A B C Citric acid 15.0 Citrate 4.0 29.0 15.0Acrylate/methacrylate 6.0 6.0 copolymer Acrylic acid maleic acid 3.7copolymer Dry add carbonate 9.0 20.0 Alkali metal silicate 8.5 17.0 9.0Paraffin 0.5 Benzotriazole 0.3 Termamyl 60T 1.5 1.5 1.0 Protease #4(4.6% prill) 1.6 1.6 1.6 Percarbonate (AvO) 1.5 Perborate monohydrate0.3 1.5 Perborate tetrahydrate 0.9 Tetraacetylethylene diamine 3.8 4.4Diethylene triamine penta 0.13 0.13 0.13 methyphosphonic acid (Mg salt)Alkyl ethoxy sulphate-3x 3.0 ethoxylated Alkyl ethoxy propoxy nonionicsurfactant Suds suppressor 2.0 Olin SLF18 nonionic surfactant Sulfate

In the immediately above formulations a reduced immunogenic protease(e.g., a variant subtilisin Carlsberg enzyme) useful in the presentinvention is substituted therein with substantially similar results.Also in the immediately above formulations, any combination of theproteases useful in the present invention (e.g., variant subtilisinCarlsberg enzymes) recited herein can be substituted in withsubstantially similar results.

3. Fabric Cleaning Compositions

The present invention further provides fabric cleaning compositionscomprising one or more protease enzymes (e.g., variant subtilisinCarlsberg enzymes).

a. Granular Fabric Cleaning

The granular fabric cleaning compositions of the present inventioncontain an effective amount of one or more protease enzymes (e.g.,variant subtilisin Carlsberg enzymes), preferably from about 0.001% toabout 10%, more preferably from about 0.005% to about 5%, morepreferably from about 0.01% to about 1% by weight of active proteaseenzyme of the composition. In addition to one or more protease enzymes,the granular fabric cleaning compositions typically comprise at leastone surfactant, one or more builders, and, in some cases, a bleachingagent. Granular fabric cleaning composition embodiments of the presentinvention are illustrated by the following examples. Granular FabricCleaning Compositions Example No. Component 20 21 22 23 Protease (4%Prill) 0.10 0.20 0.03 0.05 Protease (4% Prill) 0.02 0.05 C₁₃ linearalkyl 22.0 22.0 22.0 22.0 benzene sulfonate Phosphate 23.0 23.0 23.023.0 (as sodium tripoly- phosphates) Sodium carbonate 23.0 23.0 23.023.0 Sodium silicate 12.0 14.0 14.0 14.0 Zeolite 8.20 8.20 8.20 8.20Chelant 0.40 0.40 0.40 0.40 (diethylaenetriamine- pentaacetic acid)Sodium sulfate 5.50 5.50 5.50 5.50 Water Balance to 100%

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteases(e.g., variant subtilisin Carlsberg enzymes) useful in the presentinvention recited herein can be substituted in with substantiallysimilar results. Granular Fabric Cleaning Composition Example No.Component 24 25 26 27 Protease #(4% Prill) 0.10 0.20 0.03 0.05 Protease# +1 0.02 0.05 (4% Prill) C₁₂ alkyl benzene 12.0 12.0 12.0 12.0sulfonate Zeolite A (1-10 μm) 26.0 26.0 26.0 26.0 2-butyl octanoic 4.04.0 4.0 4.0 acid C₁₂-C₁₄ secondary 5.0 5.0 5.0 5.0 (2, 3) Sodium citrate5.0 5.0 5.0 5.0 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate17.0 17.0 17.0 17.0 Fillers, water, Balance to 100% minors

In the immediately above formulations a reduced immunogenic protease(e.g., variant subtilisin Carlsberg) useful in the present invention issubstituted therein with substantially similar results. Also in theimmediately above formulations, any combination of the proteases usefulin the present invention (e.g., variant subtilisin Carlsberg enzymes)can be substituted in with substantially similar results. GranularFabric Cleaning Compositions Example No. Component 28 29 Linear alkylbenzene sulphonate 11.4 10.7 Tallow alkyl sulphate 1.8 2.4 C₁₄₋₁₅ alkylsulphate 3.0 3.10 C₁₄₋₁₅ alcohol 7 times ethoxylated 4.0 4.0 Tallowalcohol 11 times 1.8 1.8 ethoxylated Dispersant 0.07 0.1 Silicone fluid0.80 0.80 Trisodium citrate 14.0 15.0 Citric acid 3.0 2.5 Zeolite 32.532.1 Maleic acid acrylic acid 5.0 5.9 copolymer Diethylene triaminepenta 1.0 0.20 methylene Protease # (4% Prill) 0.30 0.30 Lipase 0.360.40 Amylase 0.30 0.30 Sodium silicate 2.0 2.5 Sodium sulphate 3.5 5.2Polyvinyl pyrrolidone 0.3 0.5 Perborate 0.5 1 Phenol sulphonate 0.1 0.2Peroxidase 0.1 0.1 Minors Up to 100

Granular Fabric Cleaning Compositions Example No. Component 30 31 Sodiumlinear C₁₂ alkyl benzene 6.5 8.0 sulphonate Sodium sulphate 15.0 18.0Zeolite 26.0 22.0 Sodium nitrilotriacetate 5.0 5.0 Polyvinyl pyrrolidone0.5 0.7 Tetraacetylethylene diamine 3.0 3.0 Boric acid 4.0 Perborate 0.51 Phenol sulphonate 0.1 0.2 Protease #4 (4% Prill) 0.4 0.4 Fillers(e.g., silicates, carbonates, Up to 100 perfumes)

In additional embodiments, compact granular fabric cleaning compositionssuch as the following are provided. The ingredients are provided interms of the weight percent. Composition 1: alkyl sulphate (8.0), alkylethoxy sulphate (2.0), a mixture of C25 and C45 alcohol 3 and 7 timesethoxylated (6.0), polyhydroxy fatty acid amide (2.5), Zeolite (17.0),layered silicate/citrate (16.0), carbonate (7.0), maleic acid acrylicacid copolymer (5.0), soil release polymer (0.4), carboxymethylcellulose (0.4), poly(4-vinylpyridine)-N-oxide (0.1), copolymer ofvinylimidazole and vinylpyrrolidone (0.1), PEG-2000 (0.2), protease #(4% Prill) (0.5), lipase (0.2), cellulase (0.2), tetracetylethylenediamine (6.0), percarbonate (22.0), ethylene diamine disuccinic acid(0.3), suds suppressor (3.5),disodium-4,4′-bis(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate(0.25), Disodium-4,4′-bis(2-sulfostyril)biphenyl (0.05), and acombination of water, perfume and minors (up to 100).

In an alternative granular fabric cleaning composition, the followingingredients are provided. The ingredients are provided in terms of theweight percent. Composition 2: linear alkyl benzene sulphonate (7.6),C₁₆-C₁₈ alkyl sulfate (1.3), C₁₄₋₁₅ alcohol 7 times ethoxyiated (4.0),coco-alkyl-dimethyl hydroxyethyl ammonium chloride (1.4), dispersant(0.07), silicone fluid (0.8), trisodium citrate (5.0), Zeolite 4A(15.0), maleic acid acrylic acid copolymer (4.0), diethylene triaminepenta methylene phosphonic acid (0.4), perborate (15.0),tetraacetylethylene diamine (5.0), smectite clay (10.0), poly(oxyethylene) (MW 300,000) (0.3), protease # (4% Prill) (0.4), lipase (0.2),amylase (0.3), cellulase (0.2), sodium silicate (3.0), sodium carbonate(10.0), carboxymethyl cellulose (0.2), brighteners (0.2), and a mixtureof water, perfume and minors (up to 100).

In yet another alternative granular fabric cleaning composition, thefollowing ingredients are provided. The ingredients are provided interms of the weight percent. Composition 2: linear alkyl benzene (6.92),tallow alkyl sulfate (2.05), C₁₄₋₁₅ alcohol 7 times ethoxylated (4.4),C₁₂₋₁₅ alkyl ethoxy sulfate—3 times ethoxylated (0.16), Zeolite (20.2),citrate (5.5), carbonate (15.4), silicate (3.0), maleic acid acrylicacid copolymer (4.0), carboxymethyl cellulase (0.31), soil releasepolymer (0.30), protease # (4% Prill) (0.2), lipase (0.36), cellulase(0.13), perborate tetrahydrate (11.64), perborate monohydrate (8.7),tetraacetylethylene diamine (5.0), diethylene tramine penta methylphosphonic acid (0.38), magnesium sulfate (0.40), brightener (0.19), amixture of perfume, silicone, and suds suppressors (0.85), and minors(up to 100).

In the immediately above formulations a reduced immunogenic protease(e.g., a variant subtilisin Carlsberg enzyme) useful in the presentinvention is substituted therein with substantially similar results.Also in the immediately above formulations, any combination of theproteases useful in the present invention (e.g., variant subtilisinCarlsberg enzymes) can be substituted in with substantially similarresults.

b. Liquid Fabric Cleaning Compositions

Liquid fabric cleaning compositions of the present invention comprise aneffective amount of one or more protease enzymes (e.g., variantsubtilisin Carlsberg enzymes), preferably from about 0.0001% to about10%, more preferably from about 0.001% to about 1%, and most preferablyfrom about 0.001% to about 0.1%, by weight of active protease enzyme ofthe composition. Such liquid fabric cleaning compositions typicallyadditionally comprise an anionic surfactant, a fatty acid, awater-soluble detergency builder and water. The liquid fabric cleaningcomposition embodiment of the present invention is illustrated by thefollowing examples. Liquid Fabric Cleaning Compositions Example No.Component 35 36 37 38 39 Protease # 0.05 0.03 0.30 0.03 0.10 Protease #+1 0.01 0.20 C₁₂-C₁₄ alkyl sulfate, 20.0 20.0 20.0 20.0 20.0 Na 2-butyloctanoic acid 5.0 5.0 5.0 5.0 5.0 Sodium citrate 1.0 1.0 1.0 1.0 1.0 C₁₀alcohol 13.0 13.0 13.0 13.0 13.0 ethoxylate (3) Monethanolamine 2.5 2.52.5 2.5 2.5 Water/propylene Balance to 100% (100:1:1) glycol/ethanol

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.Liquid Fabric Cleaning Compositions Example No. Component 40 41 C₁₂₋₁₄alkyl succinic acid 3.0 8.0 Citric acid monohydrate 10.0 15.0 SodiumC₁₂₋₁₅ alkyl sulphate 8.0 8.0 Sodium sulfate of C₁₂₋₁₅ alcohol 2 3.0times ethoxylated C₁₂₋₁₅ alcohol 7 times ethoxylated 8.0 C₁₂₋₁₅ alcohol5 times ethoxylated 8.0 Diethylene triamine penta 0.2 (methylenephosphonic acid) Oleic acid 1.8 Ethanol 4.0 4.0 Propanediol 2.0 2.0Protease # 0.2 0.2 Polyvinyl pyrrolidone 1.0 2.0 Suds suppressor 0.150.15 NaOH up to pH 7.5 Perborate 0.5 1.0 Phenol sulphonate 0.1 0.2Peroxidase 0.4 0.1 Water and minors Up to 100

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.

c. Bar Fabric Cleaning Compositions

Bar fabric cleaning compositions of the present invention suitable forhand-washing soiled fabrics contain an effective amount of one or moreprotease enzymes (e.g., variant subtilisin Carlsberg enzymes),preferably from about 0.001% to about 10%, more preferably from about0.01% to about 1% by weight of the composition. The bar fabric cleaningcomposition embodiments of the present invention is illustrated by thefollowing examples. Bar Fabric Cleaning Composition Example No.Component 42 43 44 45 Protease # 0.3 0.1 0.02 Protease # +1 0.4 0.03C₁₂-C₁₆ alkyl sulfate, 20.0 20.0 20.0 20.0 Na C₁₂-C₁₄-N-methyl 5.0 5.05.0 5.0 glucamide C₁₁-C₁₃ alkyl benzene 10.0 10.0 10.0 10.0 sulfonate,Na Sodium carbonate 25.0 25.0 25.0 25.0 Sodium pyrophosphate 7.0 7.0 7.07.0 Sodium 7.0 7.0 7.0 7.0 tripolyphosphate Zeolite A 5.0 5.0 5.0 5.0(0.1-10 μm) Carboxymethylcellulose 0.2 0.2 0.2 0.2 Polyacrylate 0.2 0.20.2 0.2 (MW 1400) Coconut 5.0 5.0 5.0 5.0 monethanolamide Brightener,perfume 0.2 0.2 0.2 0.2 CaSO₄ 1.0 1.0 1.0 1.0 MgSO₄ 1.0 1.0 1.0 1.0Water 4.0 4.0 4.0 4.0 Fillers (e.g., CaCO3, Balance to 100% talc, clay,silicates, etc.)

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.

B. Additional Cleaning Compositions

In addition to the hard surface cleaning, dishwashing and fabriccleaning compositions discussed above, one or more protease enzymes(e.g., variant subtilisin Carlsberg enzymes) find use as components ofvarious other cleaning compositions where hydrolysis of an insolublesubstrate is desired. Such additional cleaning compositions include, butare not limited to oral cleaning compositions, denture cleaningcompositions, and contact lens cleaning compositions, as well as otherpersonal care cleaning compositions.

1. Oral Cleaning Compositions

In additional embodiments of the present invention,pharmaceutically-acceptable amounts of one or more protease enzymes(e.g., variant subtilisin Carlsberg enzymes) are included incompositions useful for removing proteinaceous stains from teeth ordentures. Preferably, oral cleaning compositions of the presentinvention comprise from about 0.0001% to about 20% of one or moreprotease enzymes, more preferably from about 0.001% to about 10%, morepreferably still from about 0.01% to about 5%, by weight of thecomposition, and a pharmaceutically-acceptable carrier. Typically, thepharmaceutically-acceptable oral cleaning carrier components of the oralcleaning components of the oral cleaning compositions will generallycomprise from about 50% to about 99.99%, preferably from about 65% toabout 99.99%, more preferably from about 65% to about 99%, by weight ofthe composition.

The pharmaceutically-acceptable carrier components and optionalcomponents which may be included in the oral cleaning compositions ofthe present invention are well known to those skilled in the art. A widevariety of composition types, carrier components and optional componentsuseful in the oral cleaning compositions are disclosed in U.S. Pat. No.5,096,700; U.S. Pat. No. 5,028,414; and U.S. Pat. No. 5,028,415, all ofwhich are incorporated herein by reference. Oral cleaning compositionembodiments of the present invention are illustrated by the followingexamples. Oral Dentrifice Cleaning Composition Example No. Component 4647 48 49 Protease # 2.0 3.5 1.5 2.0 Sorbitol (70% aq. 35.0 35.0 35.035.0 soln.) PEG-6* 1.0 1.0 1.0 1.0 Silica dental 20.0 20.0 20.0 20.0abrasive** Sodium fluoride 0.243 0.243 0.243 0.243 Titanium oxide 0.50.5 0.5 0.5 Sodium saccharin 0.286 0.286 0.286 0.286 Sodium alkylsulfate 4.0 4.0 4.0 4.0 (27.9%) Flavor 1.04 1.04 1.04 1.04 Carboxyvinyl0.30 0.30 0.30 0.30 polymer*** Carrageenan**** 0.8 0.8 0.8 0.8 WaterBalance to 100%*PEG-6 - Polyethylene glycol, having MW of 600**recipitated silica identified as Zeodent 119 (J. M. Huber).***Carbopol (B. F. Goodrich Chemical Co.)****lota carrageenan (Hercules Chemical Co.).

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.Mouthwash Compositions Example No. Component 50 51 52 53 Protease # 3.07.5 1.0 5.0 SDA 40 Alcohol 8.0 8.0 8.0 8.0 Flavor 0.08 0.08 0.08 0.08Emulsifier 0.08 0.08 0.08 0.08 Sodium fluoride 0.05 0.05 0.05 0.05Glycerin 10.0 10.0 10.0 10.0 Sweetener 0.02 0.02 0.02 0.02 Benzoic acid0.05 0.05 0.05 0.05 NaOH 0.20 0.20 0.20 0.20 Dye 0.04 0.04 0.04 0.04Water Balance to 100%

In the immediately above formulations, a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.Lozenge Compositions Example No. Component 54 55 56 57 Protease # 0.010.03 0.10 0.02 Sorbitol 17.50 17.50 17.50 17.50 Mannitol 17.50 17.5017.50 17.50 Starch 13.60 13.60 13.60 13.60 Sweetener 1.20 1.20 1.20 1.20Flavor 11.7 11.7 11.7 11.7 Color 0.10 0.10 0.10 0.10 Corn syrup Balanceto 100%

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.Chewing Gum Compositions Example No. Component 58 59 60 61 Protease #0.03 0.02 0.10 0.05 Sorbitol crystals 38.44 38.4 38.4 38.4 Paloja-T gumbase* 20.0 20.0 20.0 20.0 Sorbitol (70% aq. soln.) 22.0 22.0 22.0 22.0Mannitol 10.0 10.0 10.0 10.0 Glycerine 7.56 7.56 7.56 7.56 Flavor 1.01.0 1.0 1.0 Corn syrup Balance to 100%*Supplied by L.A. Dreyfus Co.

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.

2. Denture Cleaning Compositions

In yet additional embodiments, the present invention provides variousdenture cleaning compositions for cleaning dentures outside of the oralcavity comprise one or more protease enzymes (e.g., variant subtilisinCarlsberg enzymes). Such denture cleaning compositions comprise aneffective amount of one or more protease enzymes (e.g., variantsubtilisin Carlsberg enzymes), preferably from about 0.0001% to about50% is of one or more protease enzymes, more preferably from about0.001% to about 35%, more preferably still from about 0.01% to about20%, by weight of the composition, and a denture cleansing carrier.Various denture cleansing composition formats such as effervescenttablets and the like are well known in the art (See e.g., U.S. Pat. No.5,055,305, incorporated herein by reference), and are generallyappropriate for incorporation of one or more protease enzymes forremoving proteinaceous stains from dentures.

The denture cleaning composition embodiments of the present invention isillustrated by the following examples. Two-Layer Effervescent DentureCleansing Table Composition Example No. Component 62 63 64 65 AcidicLayer: Protease # 1.0 1.5 0.01 0.05 Tartaric acid 24.0 24.0 24.0 24.0Sodium carbonate 4.0 4.0 4.0 4.0 Sulphamic acid 10.0 10.0 10.0 10.0 PEG20,000 4.0 4.0 4.0 4.0 Sodium bicarbonate 24.5 24.5 24.5 24.5 Potassiumpersulfate 15.0 15.0 15.0 15.0 Sodium acid 7.0 7.0 7.0 7.0 pyrophosphatePyrogenic silica 2.0 2.0 2.0 2.0 Tetracetylethylene 7.0 7.0 7.0 7.0diamine Ricinoleylsulfosuccinate 0.5 0.5 0.5 0.5 Flavor 1.0 1.0 1.0 1.0Alkaline Layer: Sodium perborate 32.0 32.0 32.0 32.0 monohydrate Sodiumbicarbonate 19.0 19.0 19.0 19.0 EDTA 3.0 3.0 3.0 3.0 Sodiumtripolyphosphate 12.0 12.0 12.0 12.0 PEG 20,000 2.0 2.0 2.0 2.0Potassium persulfate 26.0 26.0 26.0 26.0 Sodium carbonate 2.0 2.0 2.02.0 Pyrogenic silica 2.0 2.0 2.0 2.0 Dye/flavor 2.0 2.0 2.0 2.0

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.

3. Personal Cleansing Compositions

In additional embodiments of the present invention, personal cleaningcompositions for cleaning the skin comprise one or more of the proteaseenzymes (e.g., variant subtilisin Carlsberg enzymes). Such compositionstypically comprise from about 0.001% to about 5% protease enzyme (e.g.,variant subtilisin Carlsberg enzymes), preferably from about 0.005% toabout 2%, and most preferably from about 0.01% to about 0.8% by weightof the composition. Preferred personal cleansing compositions into whichcan be included protease enzymes as described herein include, but arenot limited to those described in U.S. patent application Ser. Nos.08/121,623 and 08/121,624. Although various compositions arecontemplated by the present invention, one liquid personal cleaningcomposition containing a soap component includes (weight %): soap (K orNa) (15.00), 30% laurate, 30% myristate, 25% palmitate, 15% stearate,fatty acids (above ratios) (4.50), Na lauryl sarcosinate (6.00), sodiumlaureth-3 sulfate (0.66), cocamidopropylbetaine (1.33), glycerine(15.00), propylene glycol (9.00), polyquaternium 10 (0.80), ethyleneglycol distearate (EDTA) (1.50), propylparaben (0.10), methylparaben(0.20), protease# (0.10), KOH or NaOH (if necessary to adjust pH),calcium sulfate (3), acetic acid (3), and water (balance to 100).

In another embodiment, personal cleansing bars are provided by thepresent invention. Although various compositions are contemplated by thepresent invention, one bar personal cleaning composition containing asoap component includes (weight sodium cocoyl isethionate (47.20),sodium cetearyl sulfate (9.14), paraffin (9.05), sodium soap (in situ)(3.67), sodium isethionate (5.51), sodium chloride (0.45), titaniumdioxide (0.4), trisodium EDTA (0.1), trisodium etidronate (0.1), perfume(1.20), Na₂ SO₄ (0.87), protease # (0.10), and a mixture of water andminors (balance to 100).

In the immediately above formulations a reduced immunogenic proteaseuseful in the present invention (e.g., a variant subtilisin Carlsbergenzyme) is substituted therein with substantially similar results. Alsoin the immediately above formulations, any combination of the proteasesuseful in the present invention (e.g., variant subtilisin Carlsbergenzymes) can be substituted in with substantially similar results.

EXAMPLE 12 Wash Performance Test

The wash performance of the variants useful in the present inventioncompositions may be evaluated by any suitable means known in the art.One suitable method for measuring the removal of stain from EMPA 116(blood/milk/carbon black on cotton) cloth swatches (Testfabrics, Inc.,Middlesex, N.J. 07030) is described in this Example.

Six EMPA 116 swatches, cut to 3.times.4½ inches with pinked edges, areplaced in each pot of a Model 7243S Terg-O-Tometer (United StatesTesting Co., Inc., Hoboken, N.J.) containing 1000 ml of water, 15 gpghardness (Ca++:Mg++::3:1::w:w), 7 g of detergent, and enzyme asappropriate. The detergent base is WFK1 detergent from wfk—TestgewebeGmbH, (Krefeld, Germany) and has the following components (% of finalformulation): Zeolite A (25%), sodium sulfate (25%), soda ash (10%),linear alkylbenzenesulfonate (8.8%), alcohol ethoxylate (7-8 EO) (4.5%),sodium soap (3%), and sodium silicate (SiO₂:Na₂O::3.3:1)(3%).

To this base detergent, the following additions are made (% of finalformulation): sodium perborate monohydrate (13%), copolymer (SokalanCP5) (4%), TAED (Mykon ATC Green) (3%), enzyme (0.5%), and whitener(Tinopal AMS-GX) (0.2%).

Sodium perborate monohydrate can be obtained from various commercialsources, including Degussa Corporation, Ridgefield-Park. Sokalan CP5 isobtained from BASF Corporation, Parsippany, N.J. Mykon ATC Green (TAED,tetraacetylethylenediamine) can be obtained from Warwick International,Limited, England. T inopal AMS GX can be obtained from Ciba-GeigyCorporation, Greensboro, N.C.

In one suitable testing method, six EMPA 116 swatches are washed indetergent with enzyme for 30 min at 60° C., rinsed twice for 5 minuteseach time in 1000 ml water. Enzymes are added at final concentrations of0.05 to 1 ppm for standard curves, and 0.25 ppm for routine analyses.Swatches are dried and pressed, and the reflectance from the swatches ismeasured using the L value on the L*a*b* scale of a Minolta ChromaMeter, Model CR-200 (Minolta Corporation, Ramsey, N.J.). In someembodiments, the performance of the test enzyme is reported as apercentage of the performance of B. amyloliquefaciens (BPN′) proteaseand is calculated by dividing the amount of B. amyloliquefaciens (BPN′)protease by the amount of variant protease (e.g., variant subtilisinCarlsberg enzyme) that is needed to provide the same stain removalperformance.times.100.

EXAMPLE 13 Variant Subtilisin Carlsberg Stability in a Liquid DetergentFormulation

This example provides a means for comparison of protease (e.g., variantsubtilisin Carlsberg enzyme) stability toward inactivation in a liquiddetergent formulation with Bacillus amyloliquefaciens subtilisin and itsvariant enzymes. As other methods find use with the present invention,it is not intended that the present invention be limited to this method.

In this method, the detergent formulation for the study is acommercially available laundry detergent (e.g., Tide® Ultra liquidlaundry detergent (Proctor & Gamble)). In some embodiments, heattreatment of the detergent formulation is necessary to inactivatein-situ protease. This is accomplished by incubating the detergent at96° C. for a period of 4.5 hours. Concentrated preparations of the B.amyloliquefaciens subtilisin and variant (e.g., variant subtilisinCarisberg enzymes) to be tested, in the range of 20 grams/liter enzyme,are then added to the heat-treated detergent, at room-temperature to afinal concentration of 0.3 grams/liter enzyme in the detergentformulation. The heat-treated detergent with protease added is thenincubated in a water bath at 50° C. Aliquots are removed from theincubation tubes at 0, 24, 46, 76, and 112 hour time intervals andassayed for enzyme activity by addition to a 1 cm cuvette containing 1.2mM of the synthetic peptide substrate suc-Ala-Ala-Pro-phe-p-nitroanilidedissolved in 0.1 M Tris-HCL buffer, pH 8.6, and at 25° C. The initiallinear reaction velocity is followed spectrophotometrically bymonitoring the absorbance of the reaction product p-nitroaniline at 410nm as a function of time. In preferred embodiments, the preferredvariant(s) are observed to have significantly greater stability towardsinactivation than the native B. amyloliquefaciens enzyme. Estimatedhalf-lives for inactivation in the laundry detergent formulation for thetwo enzymes are determined under the specified test conditions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which that are obvious to those skilled in molecularbiology, immunology, formulations, and/or related fields are intended tobe within the scope of the present invention.

1. A method for identifying at least one T-cell epitope of a microbialsubtilisin, wherein said subtilisin comprises subtilisin Carlsberg,comprising the steps of: (i) obtaining from a from a single human bloodsource, a solution of dendritic cells and a solution of naïve CD4+and/or CD8+ T-cells; (ii) differentiating said dendritic cells toproduce a solution of differentiated dendritic cells; (iii) combiningsaid solution of differentiated dendritic cells and said naïve CD4+and/or CD8+ T-cells with peptide fragments of said subtilisin Carlsberg;and (iv) measuring proliferation of said T-cells in said step (iii). 2.The method of claim 1, wherein said microbial subtilisin Carlsberg isderived from a member of the genus Bacillus.
 3. The method of claim 2,wherein the Bacillus is selected from the group consisting of B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.4. The method of claim 1, wherein said microbial subtilisin Carlsbergcomprises at least a portion of the sequence set forth in SEQ ID NO:1.5. A method of reducing the immunogenicity of a microbial subtilisinCarlsberg, comprising the steps of: (a) identifying at least one T-cellepitope in said protein by (i) contacting an adherent monocyte-deriveddendritic cell that has been differentiated by exposure to at least onecytokine in vitro, with at least one peptide comprising said T-cellepitope; and (ii) contacting said dendritic cell and said peptide with anaïve T-cell, wherein said naïve T-cell has been obtained from the samesource as said adherent monocyte-derived dendritic cell, and wherebysaid T-cell proliferates in response to said peptide; and (b) modifyingsaid subtilisin Carlsberg to neutralize said T-cell epitope to produce avariant protein, such that said variant protein induces less than orsubstantially equal to the baseline proliferation of said naïve T-cells.6. The method of claim 5, wherein said microbial subtilisin is derivedfrom a member of the genus Bacillus.
 7. The method of claim 6, whereinthe Bacillus is selected from the group consisting of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.8. The method of claim 5, wherein said microbial subtilisin Carlsbergcomprises at least a portion of the sequence set forth in SEQ ID NO:1.9. The method of claim 5, said epitope of said microbial subtilisinCarlsberg is modified by: (a) substituting the amino acid sequence ofsaid T-cell epitope with an analogous sequence from a homolog of saidmicrobial subtilisin, wherein said substitution substantially mimics themajor tertiary structure attributes of the T-cell epitope.
 10. Themethod of claim 5, wherein said microbial subtilisin Carlsberg ismodified by altering at least one epitope selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:90, SEQ ID NO:15, SEQ ID NO:30, andSEQ ID NO:40.
 11. The method of claim 10, wherein said epitope ismodified by substituting an amino acid sequence for a residuecorresponding to at least one of said epitopes.
 12. The method of claim10, wherein said epitope is modified by deleting an amino acid sequencefor a residue corresponding to at least one of said epitopes.
 13. Themethod of claim 10, wherein said epitope is modified by adding an aminoacid to at least one of said epitopes.
 14. A modified subtilisinCarlsberg, wherein said subtilisin Carlsberg comprises at least onealteration in at least one epitope comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:90, SEQ IDNO:15, SEQ ID NO:30, and SEQ ID NO:40.
 15. The modified subtilisinCarlsberg of claim 14, wherein said modified subtilisin Carlsberg isexpressed in an organism within the genus Bacillus.
 16. The modifiedsubtilisin Carlsberg of claim 14, wherein the immunogenic responseproduced by said modified subtilisin Carlsberg is less than saidimmunogenic response produced by wild-type modified subtilisinCarlsberg.
 17. The modified subtilisin Carlsberg of claim 14, whereinthe immunogenic response produced by said modified subtilisin Carlsbergis greater than said immunogenic response produced by wild-type modifiedsubtilisin Carlsberg.
 18. A composition comprising nucleic acid encodingsaid modified subtilisin Carlsberg of claim
 14. 19. An expression vectorcomprising the nucleic acid of claim
 18. 20. A host cell transformedwith the expression vector of claim
 19. 21. A composition selected fromthe group consisting of cleaning compositions, personal carecompositions, and healthcare compositions, comprising the modifiedsubtilisin Carlsberg of claim 14.